Real-time Image Processing for Microscopy-based Label-free Imaging Flow Cytometry in a Microfluidic Chip.

PubMed

Heo, Young Jin; Lee, Donghyeon; Kang, Junsu; Lee, Keondo; Chung, Wan Kyun

2017-09-14

Imaging flow cytometry (IFC) is an emerging technology that acquires single-cell images at high-throughput for analysis of a cell population. Rich information that comes from high sensitivity and spatial resolution of a single-cell microscopic image is beneficial for single-cell analysis in various biological applications. In this paper, we present a fast image-processing pipeline (R-MOD: Real-time Moving Object Detector) based on deep learning for high-throughput microscopy-based label-free IFC in a microfluidic chip. The R-MOD pipeline acquires all single-cell images of cells in flow, and identifies the acquired images as a real-time process with minimum hardware that consists of a microscope and a high-speed camera. Experiments show that R-MOD has the fast and reliable accuracy (500 fps and 93.3% mAP), and is expected to be used as a powerful tool for biomedical and clinical applications.

Metabolic Imaging in Multiple Time Scales

PubMed Central

Ramanujan, V Krishnan

2013-01-01

We report here a novel combination of time-resolved imaging methods for probing mitochondrial metabolism multiple time scales at the level of single cells. By exploiting a mitochondrial membrane potential reporter fluorescence we demonstrate the single cell metabolic dynamics in time scales ranging from milliseconds to seconds to minutes in response to glucose metabolism and mitochondrial perturbations in real time. Our results show that in comparison with normal human mammary epithelial cells, the breast cancer cells display significant alterations in metabolic responses at all measured time scales by single cell kinetics, fluorescence recovery after photobleaching and by scaling analysis of time-series data obtained from mitochondrial fluorescence fluctuations. Furthermore scaling analysis of time-series data in living cells with distinct mitochondrial dysfunction also revealed significant metabolic differences thereby suggesting the broader applicability (e.g. in mitochondrial myopathies and other metabolic disorders) of the proposed strategies beyond the scope of cancer metabolism. We discuss the scope of these findings in the context of developing portable, real-time metabolic measurement systems that can find applications in preclinical and clinical diagnostics. PMID:24013043

Single-Cell and Single-Molecule Analysis of Gene Expression Regulation.

PubMed

Vera, Maria; Biswas, Jeetayu; Senecal, Adrien; Singer, Robert H; Park, Hye Yoon

2016-11-23

Recent advancements in single-cell and single-molecule imaging technologies have resolved biological processes in time and space that are fundamental to understanding the regulation of gene expression. Observations of single-molecule events in their cellular context have revealed highly dynamic aspects of transcriptional and post-transcriptional control in eukaryotic cells. This approach can relate transcription with mRNA abundance and lifetimes. Another key aspect of single-cell analysis is the cell-to-cell variability among populations of cells. Definition of heterogeneity has revealed stochastic processes, determined characteristics of under-represented cell types or transitional states, and integrated cellular behaviors in the context of multicellular organisms. In this review, we discuss novel aspects of gene expression of eukaryotic cells and multicellular organisms revealed by the latest advances in single-cell and single-molecule imaging technology.

Single Vector Calibration System for Multi-Axis Load Cells and Method for Calibrating a Multi-Axis Load Cell

NASA Technical Reports Server (NTRS)

Parker, Peter A. (Inventor)

2003-01-01

A single vector calibration system is provided which facilitates the calibration of multi-axis load cells, including wind tunnel force balances. The single vector system provides the capability to calibrate a multi-axis load cell using a single directional load, for example loading solely in the gravitational direction. The system manipulates the load cell in three-dimensional space, while keeping the uni-directional calibration load aligned. The use of a single vector calibration load reduces the set-up time for the multi-axis load combinations needed to generate a complete calibration mathematical model. The system also reduces load application inaccuracies caused by the conventional requirement to generate multiple force vectors. The simplicity of the system reduces calibration time and cost, while simultaneously increasing calibration accuracy.

Single-cell transcriptome conservation in cryopreserved cells and tissues.

PubMed

Guillaumet-Adkins, Amy; Rodríguez-Esteban, Gustavo; Mereu, Elisabetta; Mendez-Lago, Maria; Jaitin, Diego A; Villanueva, Alberto; Vidal, August; Martinez-Marti, Alex; Felip, Enriqueta; Vivancos, Ana; Keren-Shaul, Hadas; Heath, Simon; Gut, Marta; Amit, Ido; Gut, Ivo; Heyn, Holger

2017-03-01

A variety of single-cell RNA preparation procedures have been described. So far, protocols require fresh material, which hinders complex study designs. We describe a sample preservation method that maintains transcripts in viable single cells, allowing one to disconnect time and place of sampling from subsequent processing steps. We sequence single-cell transcriptomes from >1000 fresh and cryopreserved cells using 3'-end and full-length RNA preparation methods. Our results confirm that the conservation process did not alter transcriptional profiles. This substantially broadens the scope of applications in single-cell transcriptomics and could lead to a paradigm shift in future study designs.

CellStress - open source image analysis program for single-cell analysis

NASA Astrophysics Data System (ADS)

Smedh, Maria; Beck, Caroline; Sott, Kristin; Goksör, Mattias

2010-08-01

This work describes our image-analysis software, CellStress, which has been developed in Matlab and is issued under a GPL license. CellStress was developed in order to analyze migration of fluorescent proteins inside single cells during changing environmental conditions. CellStress can also be used to score information regarding protein aggregation in single cells over time, which is especially useful when monitoring cell signaling pathways involved in e.g. Alzheimer's or Huntington's disease. Parallel single-cell analysis of large numbers of cells is an important part of the research conducted in systems biology and quantitative biology in order to mathematically describe cellular processes. To quantify properties for single cells, large amounts of data acquired during extended time periods are needed. Manual analyses of such data involve huge efforts and could also include a bias, which complicates the use and comparison of data for further simulations or modeling. Therefore, it is necessary to have an automated and unbiased image analysis procedure, which is the aim of CellStress. CellStress utilizes cell contours detected by CellStat (developed at Fraunhofer-Chalmers Centre), which identifies cell boundaries using bright field images, and thus reduces the fluorescent labeling needed.

Time-resolved, single-cell analysis of induced and programmed cell death via non-invasive propidium iodide and counterstain perfusion.

PubMed

Krämer, Christina E M; Wiechert, Wolfgang; Kohlheyer, Dietrich

2016-09-01

Conventional propidium iodide (PI) staining requires the execution of multiple steps prior to analysis, potentially affecting assay results as well as cell vitality. In this study, this multistep analysis method has been transformed into a single-step, non-toxic, real-time method via live-cell imaging during perfusion with 0.1 μM PI inside a microfluidic cultivation device. Dynamic PI staining was an effective live/dead analytical tool and demonstrated consistent results for single-cell death initiated by direct or indirect triggers. Application of this method for the first time revealed the apparent antibiotic tolerance of wild-type Corynebacterium glutamicum cells, as indicated by the conversion of violet fluorogenic calcein acetoxymethyl ester (CvAM). Additional implementation of this method provided insight into the induced cell lysis of Escherichia coli cells expressing a lytic toxin-antitoxin module, providing evidence for non-lytic cell death and cell resistance to toxin production. Finally, our dynamic PI staining method distinguished necrotic-like and apoptotic-like cell death phenotypes in Saccharomyces cerevisiae among predisposed descendants of nutrient-deprived ancestor cells using PO-PRO-1 or green fluorogenic calcein acetoxymethyl ester (CgAM) as counterstains. The combination of single-cell cultivation, fluorescent time-lapse imaging, and PI perfusion facilitates spatiotemporally resolved observations that deliver new insights into the dynamics of cellular behaviour.

Three-dimensional reconstruction of single-cell chromosome structure using recurrence plots.

PubMed

Hirata, Yoshito; Oda, Arisa; Ohta, Kunihiro; Aihara, Kazuyuki

2016-10-11

Single-cell analysis of the three-dimensional (3D) chromosome structure can reveal cell-to-cell variability in genome activities. Here, we propose to apply recurrence plots, a mathematical method of nonlinear time series analysis, to reconstruct the 3D chromosome structure of a single cell based on information of chromosomal contacts from genome-wide chromosome conformation capture (Hi-C) data. This recurrence plot-based reconstruction (RPR) method enables rapid reconstruction of a unique structure in single cells, even from incomplete Hi-C information.

Three-dimensional reconstruction of single-cell chromosome structure using recurrence plots

NASA Astrophysics Data System (ADS)

Hirata, Yoshito; Oda, Arisa; Ohta, Kunihiro; Aihara, Kazuyuki

2016-10-01

Single-cell analysis of the three-dimensional (3D) chromosome structure can reveal cell-to-cell variability in genome activities. Here, we propose to apply recurrence plots, a mathematical method of nonlinear time series analysis, to reconstruct the 3D chromosome structure of a single cell based on information of chromosomal contacts from genome-wide chromosome conformation capture (Hi-C) data. This recurrence plot-based reconstruction (RPR) method enables rapid reconstruction of a unique structure in single cells, even from incomplete Hi-C information.

Inferring fitness landscapes and selection on phenotypic states from single-cell genealogical data

PubMed Central

Kussell, Edo

2017-01-01

Recent advances in single-cell time-lapse microscopy have revealed non-genetic heterogeneity and temporal fluctuations of cellular phenotypes. While different phenotypic traits such as abundance of growth-related proteins in single cells may have differential effects on the reproductive success of cells, rigorous experimental quantification of this process has remained elusive due to the complexity of single cell physiology within the context of a proliferating population. We introduce and apply a practical empirical method to quantify the fitness landscapes of arbitrary phenotypic traits, using genealogical data in the form of population lineage trees which can include phenotypic data of various kinds. Our inference methodology for fitness landscapes determines how reproductivity is correlated to cellular phenotypes, and provides a natural generalization of bulk growth rate measures for single-cell histories. Using this technique, we quantify the strength of selection acting on different cellular phenotypic traits within populations, which allows us to determine whether a change in population growth is caused by individual cells’ response, selection within a population, or by a mixture of these two processes. By applying these methods to single-cell time-lapse data of growing bacterial populations that express a resistance-conferring protein under antibiotic stress, we show how the distributions, fitness landscapes, and selection strength of single-cell phenotypes are affected by the drug. Our work provides a unified and practical framework for quantitative measurements of fitness landscapes and selection strength for any statistical quantities definable on lineages, and thus elucidates the adaptive significance of phenotypic states in time series data. The method is applicable in diverse fields, from single cell biology to stem cell differentiation and viral evolution. PMID:28267748

Application of porous metal enrichment probe sampling to single cell analysis using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS).

PubMed

Fu, Qiang; Tang, Jun; Cui, Meng; Xing, Junpeng; Liu, Zhiqiang; Liu, Shuying

2016-01-01

There is an increasing need for analyzing metabolism in a single cell, which is important to understand the nature of cellular heterogeneity, disease, growth and specialization, etc. However, single cell analysis is often challenging for the traces of samples. In the present study, porous metal enrichment probe sampling combined with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) has been applied for in situ analysis of live onion epidemic cell. Porous probe, treated by corroding copper wire with HCl, was directly inserted into a single cell to get cell solution. A self-made linear actuator was enough to control the penetration of probe into the target cell accurately. Then samples on the tip of probe were eluted and detected by a commercial MALDI-TOF-MS directly. The formation of porous microstructure on the probe surface increased the adsorptive capacity of cell solution. The sensitivity of porous probe sampling was 6 times higher than uncorroded probes generally. This method provides a sensitive and convenient way for the sampling and detection of single cell solution. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Quantifying intrinsic and extrinsic control of single-cell fates in cancer and stem/progenitor cell pedigrees with competing risks analysis

PubMed Central

Cornwell, J. A.; Hallett, R. M.; der Mauer, S. Auf; Motazedian, A.; Schroeder, T.; Draper, J. S.; Harvey, R. P.; Nordon, R. E.

2016-01-01

The molecular control of cell fate and behaviour is a central theme in biology. Inherent heterogeneity within cell populations requires that control of cell fate is studied at the single-cell level. Time-lapse imaging and single-cell tracking are powerful technologies for acquiring cell lifetime data, allowing quantification of how cell-intrinsic and extrinsic factors control single-cell fates over time. However, cell lifetime data contain complex features. Competing cell fates, censoring, and the possible inter-dependence of competing fates, currently present challenges to modelling cell lifetime data. Thus far such features are largely ignored, resulting in loss of data and introducing a source of bias. Here we show that competing risks and concordance statistics, previously applied to clinical data and the study of genetic influences on life events in twins, respectively, can be used to quantify intrinsic and extrinsic control of single-cell fates. Using these statistics we demonstrate that 1) breast cancer cell fate after chemotherapy is dependent on p53 genotype; 2) granulocyte macrophage progenitors and their differentiated progeny have concordant fates; and 3) cytokines promote self-renewal of cardiac mesenchymal stem cells by symmetric divisions. Therefore, competing risks and concordance statistics provide a robust and unbiased approach for evaluating hypotheses at the single-cell level. PMID:27250534

An Optogenetic Platform for Real-Time, Single-Cell Interrogation of Stochastic Transcriptional Regulation.

PubMed

Rullan, Marc; Benzinger, Dirk; Schmidt, Gregor W; Milias-Argeitis, Andreas; Khammash, Mustafa

2018-05-17

Transcription is a highly regulated and inherently stochastic process. The complexity of signal transduction and gene regulation makes it challenging to analyze how the dynamic activity of transcriptional regulators affects stochastic transcription. By combining a fast-acting, photo-regulatable transcription factor with nascent RNA quantification in live cells and an experimental setup for precise spatiotemporal delivery of light inputs, we constructed a platform for the real-time, single-cell interrogation of transcription in Saccharomyces cerevisiae. We show that transcriptional activation and deactivation are fast and memoryless. By analyzing the temporal activity of individual cells, we found that transcription occurs in bursts, whose duration and timing are modulated by transcription factor activity. Using our platform, we regulated transcription via light-driven feedback loops at the single-cell level. Feedback markedly reduced cell-to-cell variability and led to qualitative differences in cellular transcriptional dynamics. Our platform establishes a flexible method for studying transcriptional dynamics in single cells. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Nanosecond fluorescence microscopy. Emission kinetics of fura-2 in single cells.

PubMed Central

Keating, S M; Wensel, T G

1991-01-01

A microscope based time-correlated single photon counting instrument has been constructed to measure fluorescence intensity and emission anisotropy decays from fluorophores in single cells on a nanosecond time scale. The sample is excited and the emission collected using epi-illumination optics with frequency-doubled pulses from the cavity-dumped output of a synchronously pumped dye laser serving as an excitation source. Collection of decays from a single cell is possible due to the presence of an iris in the emission path that can be reduced to less than the diameter of a single cell. Using the instrument the decay of 60 nM 1,6-diphenyl-1,3,5-hexatriene was measured, demonstrating that adequate data for lifetime analysis can be recorded from fewer 10(3) molecules of the fluorophore in an illuminated volume of 23 fl. In addition, the intensity and anisotropy decays of fura-2 in single adherent cells and in suspensions of fura-2 loaded cells in suspension, although the relative amplitudes and decay constants vary somewhat from cell to cell. The results indicate that a significant but variable fraction of fura-2 is bound to relatively immobile macromolecular components in these cells. PMID:2015383

The nature and nurture of cell heterogeneity: accounting for macrophage gene-environment interactions with single-cell RNA-Seq.

PubMed

Wills, Quin F; Mellado-Gomez, Esther; Nolan, Rory; Warner, Damien; Sharma, Eshita; Broxholme, John; Wright, Benjamin; Lockstone, Helen; James, William; Lynch, Mark; Gonzales, Michael; West, Jay; Leyrat, Anne; Padilla-Parra, Sergi; Filippi, Sarah; Holmes, Chris; Moore, Michael D; Bowden, Rory

2017-01-07

Single-cell RNA-Seq can be a valuable and unbiased tool to dissect cellular heterogeneity, despite the transcriptome's limitations in describing higher functional phenotypes and protein events. Perhaps the most important shortfall with transcriptomic 'snapshots' of cell populations is that they risk being descriptive, only cataloging heterogeneity at one point in time, and without microenvironmental context. Studying the genetic ('nature') and environmental ('nurture') modifiers of heterogeneity, and how cell population dynamics unfold over time in response to these modifiers is key when studying highly plastic cells such as macrophages. We introduce the programmable Polaris™ microfluidic lab-on-chip for single-cell sequencing, which performs live-cell imaging while controlling for the culture microenvironment of each cell. Using gene-edited macrophages we demonstrate how previously unappreciated knockout effects of SAMHD1, such as an altered oxidative stress response, have a large paracrine signaling component. Furthermore, we demonstrate single-cell pathway enrichments for cell cycle arrest and APOBEC3G degradation, both associated with the oxidative stress response and altered proteostasis. Interestingly, SAMHD1 and APOBEC3G are both HIV-1 inhibitors ('restriction factors'), with no known co-regulation. As single-cell methods continue to mature, so will the ability to move beyond simple 'snapshots' of cell populations towards studying the determinants of population dynamics. By combining single-cell culture, live-cell imaging, and single-cell sequencing, we have demonstrated the ability to study cell phenotypes and microenvironmental influences. It's these microenvironmental components - ignored by standard single-cell workflows - that likely determine how macrophages, for example, react to inflammation and form treatment resistant HIV reservoirs.

Dynamic single-cell NAD(P)H measurement reveals oscillatory metabolism throughout the E. coli cell division cycle.

PubMed

Zhang, Zheng; Milias-Argeitis, Andreas; Heinemann, Matthias

2018-02-01

Recent work has shown that metabolism between individual bacterial cells in an otherwise isogenetic population can be different. To investigate such heterogeneity, experimental methods to zoom into the metabolism of individual cells are required. To this end, the autofluoresence of the redox cofactors NADH and NADPH offers great potential for single-cell dynamic NAD(P)H measurements. However, NAD(P)H excitation requires UV light, which can cause cell damage. In this work, we developed a method for time-lapse NAD(P)H imaging in single E. coli cells. Our method combines a setup with reduced background emission, UV-enhanced microscopy equipment and optimized exposure settings, overall generating acceptable NAD(P)H signals from single cells, with minimal negative effect on cell growth. Through different experiments, in which we perturb E. coli's redox metabolism, we demonstrated that the acquired fluorescence signal indeed corresponds to NAD(P)H. Using this new method, for the first time, we report that intracellular NAD(P)H levels oscillate along the bacterial cell division cycle. The developed method for dynamic measurement of NAD(P)H in single bacterial cells will be an important tool to zoom into metabolism of individual cells.

Highly multiplexed targeted DNA sequencing from single nuclei.

PubMed

Leung, Marco L; Wang, Yong; Kim, Charissa; Gao, Ruli; Jiang, Jerry; Sei, Emi; Navin, Nicholas E

2016-02-01

Single-cell DNA sequencing methods are challenged by poor physical coverage, high technical error rates and low throughput. To address these issues, we developed a single-cell DNA sequencing protocol that combines flow-sorting of single nuclei, time-limited multiple-displacement amplification (MDA), low-input library preparation, DNA barcoding, targeted capture and next-generation sequencing (NGS). This approach represents a major improvement over our previous single nucleus sequencing (SNS) Nature Protocols paper in terms of generating higher-coverage data (>90%), thereby enabling the detection of genome-wide variants in single mammalian cells at base-pair resolution. Furthermore, by pooling 48-96 single-cell libraries together for targeted capture, this approach can be used to sequence many single-cell libraries in parallel in a single reaction. This protocol greatly reduces the cost of single-cell DNA sequencing, and it can be completed in 5-6 d by advanced users. This single-cell DNA sequencing protocol has broad applications for studying rare cells and complex populations in diverse fields of biological research and medicine.

Timing the start of division in E. coli: a single-cell study

NASA Astrophysics Data System (ADS)

Reshes, G.; Vanounou, S.; Fishov, I.; Feingold, M.

2008-12-01

We monitor the shape dynamics of individual E. coli cells using time-lapse microscopy together with accurate image analysis. This allows measuring the dynamics of single-cell parameters throughout the cell cycle. In previous work, we have used this approach to characterize the main features of single-cell morphogenesis between successive divisions. Here, we focus on the behavior of the parameters that are related to cell division and study their variation over a population of 30 cells. In particular, we show that the single-cell data for the constriction width dynamics collapse onto a unique curve following appropriate rescaling of the corresponding variables. This suggests the presence of an underlying time scale that determines the rate at which the cell cycle advances in each individual cell. For the case of cell length dynamics a similar rescaling of variables emphasizes the presence of a breakpoint in the growth rate at the time when division starts, τc. We also find that the τc of individual cells is correlated with their generation time, τg, and inversely correlated with the corresponding length at birth, L0. Moreover, the extent of the T-period, τg - τc, is apparently independent of τg. The relations between τc, τg and L0 indicate possible compensation mechanisms that maintain cell length variability at about 10%. Similar behavior was observed for both fast-growing cells in a rich medium (LB) and for slower growth in a minimal medium (M9-glucose). To reveal the molecular mechanisms that lead to the observed organization of the cell cycle, we should further extend our approach to monitor the formation of the divisome.

Chemical imaging of molecular changes in a hydrated single cell by dynamic secondary ion mass spectrometry and super-resolution microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hua, Xin; Szymanski, Craig; Wang, Zhaoying

2016-01-01

Chemical imaging of single cells is important in capturing biological dynamics. Single cell correlative imaging is realized between structured illumination microscopy (SIM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) using System for Analysis at the Liquid Vacuum Interface (SALVI), a multimodal microreactor. SIM characterized cells and guided subsequent ToF-SIMS analysis. Dynamic ToF-SIMS provided time- and space-resolved cell molecular mapping. Lipid fragments were identified in the hydrated cell membrane. Principal component analysis was used to elucidate chemical component differences among mouse lung cells that uptake zinc oxide nanoparticles. Our results provided submicron chemical spatial mapping for investigations of cell dynamics atmore » the molecular level.« less

Isolation and gene expression analysis of single potential human spermatogonial stem cells.

PubMed

von Kopylow, K; Schulze, W; Salzbrunn, A; Spiess, A-N

2016-04-01

It is possible to isolate pure populations of single potential human spermatogonial stem cells without somatic contamination for down-stream applications, for example cell culture and gene expression analysis. We isolated pure populations of single potential human spermatogonial stem cells (hSSC) without contaminating somatic cells and analyzed gene expression of these cells via single-cell real-time RT-PCR. The isolation of a pure hSSC fraction could enable clinical applications such as fertility preservation for prepubertal boys and in vitro-spermatogenesis. By utilizing largely nonspecific markers for the isolation of spermatogonia (SPG) and hSSC, previously published cell selection methods are not able to deliver pure target cell populations without contamination by testicular somatic cells. However, uniform cell populations free of somatic cells are necessary to guarantee defined growth conditions in cell culture experiments and to prevent unintended stem cell differentiation. Fibroblast growth factor receptor 3 (FGFR3) is a cell surface protein of human undifferentiated A-type SPG and a promising candidate marker for hSSC. It is exclusively expressed in small, non-proliferating subgroups of this spermatogonial cell type together with the pluripotency-associated protein and spermatogonial nuclear marker undifferentiated embryonic cell transcription factor 1 (UTF1). We specifically selected the FGFR3-positive spermatogonial subpopulation from two 30 mg biopsies per patient from a total of 37 patients with full spermatogenesis and three patients with meiotic arrest. We then employed cell selection with magnetic beads in combination with a fluorescence-activated cell sorter antibody directed against human FGFR3 to tag and visually identify human FGFR3-positive spermatogonia. Positively selected and bead-labeled cells were subsequently picked with a micromanipulator. Analysis of the isolated cells was carried out by single-cell real-time RT-PCR, real-time RT-PCR, immunocytochemistry and live/dead staining. Single-cell real-time RT-PCR and real-time RT-PCR of pooled cells indicate that bead-labeled single cells express FGFR3 with high heterogeneity at the mRNA level, while bead-unlabeled cells lack FGFR3 mRNA. Furthermore, isolated cells exhibit strong immunocytochemical staining for the stem cell factor UTF1 and are viable. The cell population isolated in this study has to be tested for their potential stem cell characteristics via xenotransplantation. Due to the small amount of the isolated cells, propagation by cell culture will be essential. Other potential hSSC without FGFR3 surface expression will not be captured with the provided experimental design. The technical approach as developed in this work could encourage the scientific community to test other established or novel hSSC markers on single SPG that present with potential stem cell-like features. The project was funded by the DFG Research Unit FOR1041 Germ cell potential (SCH 587/3-2) and DFG grants to K.v.K. (KO 4769/2-1) and A.-N.S. (SP 721/4-1). The authors declare no competing interests. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Image analysis driven single-cell analytics for systems microbiology.

PubMed

Balomenos, Athanasios D; Tsakanikas, Panagiotis; Aspridou, Zafiro; Tampakaki, Anastasia P; Koutsoumanis, Konstantinos P; Manolakos, Elias S

2017-04-04

Time-lapse microscopy is an essential tool for capturing and correlating bacterial morphology and gene expression dynamics at single-cell resolution. However state-of-the-art computational methods are limited in terms of the complexity of cell movies that they can analyze and lack of automation. The proposed Bacterial image analysis driven Single Cell Analytics (BaSCA) computational pipeline addresses these limitations thus enabling high throughput systems microbiology. BaSCA can segment and track multiple bacterial colonies and single-cells, as they grow and divide over time (cell segmentation and lineage tree construction) to give rise to dense communities with thousands of interacting cells in the field of view. It combines advanced image processing and machine learning methods to deliver very accurate bacterial cell segmentation and tracking (F-measure over 95%) even when processing images of imperfect quality with several overcrowded colonies in the field of view. In addition, BaSCA extracts on the fly a plethora of single-cell properties, which get organized into a database summarizing the analysis of the cell movie. We present alternative ways to analyze and visually explore the spatiotemporal evolution of single-cell properties in order to understand trends and epigenetic effects across cell generations. The robustness of BaSCA is demonstrated across different imaging modalities and microscopy types. BaSCA can be used to analyze accurately and efficiently cell movies both at a high resolution (single-cell level) and at a large scale (communities with many dense colonies) as needed to shed light on e.g. how bacterial community effects and epigenetic information transfer play a role on important phenomena for human health, such as biofilm formation, persisters' emergence etc. Moreover, it enables studying the role of single-cell stochasticity without losing sight of community effects that may drive it.

Detection of single photoluminescent diamond nanoparticles in cells and study of the internalization pathway.

PubMed

Faklaris, Orestis; Garrot, Damien; Joshi, Vandana; Druon, Frédéric; Boudou, Jean-Paul; Sauvage, Thierry; Georges, Patrick; Curmi, Patrick A; Treussart, François

2008-12-01

Diamond nanoparticles are promising photoluminescent probes for tracking intracellular processes, due to embedded, perfectly photostable color centers. In this work, the spontaneous internalization of such nanoparticles (diameter 25 nm) in HeLa cancer cells is investigated by confocal microscopy and time-resolved techniques. Nanoparticles are observed inside the cell cytoplasm at the single-particle and single-color-center level, assessed by time-correlation intensity measurements. Improvement of the nanoparticle signal-to-noise ratio inside the cell is achieved using a pulsed-excitation laser and time-resolved detection taking advantage of the long radiative lifetime of the color-center excited state as compared to cell autofluorescence. The internalization pathways are also investigated, with endosomal marking and colocalization analyses. The low colocalization ratio observed proves that nanodiamonds are not trapped in endosomes, a promising result in prospect of drug delivery by these nanoparticles. Low cytotoxicity of these nanoparticles in this cell line is also shown.

QUANTITATION OF INTRACELLULAR NAD(P)H IN LIVING CELLS CAN MONITOR AN IMBALANCE OF DNA SINGLE STRAND BREAK REPAIR IN REAL TIME

EPA Science Inventory

Quantitation of intracellular NAD(P)H in living cells can monitor an imbalance of DNA single strand break repair in real time.

ABSTRACT

DNA single strand breaks (SSBs) are one of the most frequent DNA lesions in genomic DNA generated either by oxidative stress or du...

Visualization of phage DNA degradation by a type I CRISPR-Cas system at the single-cell level.

PubMed

Guan, Jingwen; Shi, Xu; Burgos, Roberto; Zeng, Lanying

2017-03-01

The CRISPR-Cas system is a widespread prokaryotic defense system which targets and cleaves invasive nucleic acids, such as plasmids or viruses. So far, a great number of studies have focused on the components and mechanisms of this system, however, a direct visualization of CRISPR-Cas degrading invading DNA in real-time has not yet been studied at the single-cell level. In this study, we fluorescently label phage lambda DNA in vivo , and track the labeled DNA over time to characterize DNA degradation at the single-cell level. At the bulk level, the lysogenization frequency of cells harboring CRISPR plasmids decreases significantly compared to cells with a non-CRISPR control. At the single-cell level, host cells with CRISPR activity are unperturbed by phage infection, maintaining normal growth like uninfected cells, where the efficiency of our anti-lambda CRISPR system is around 26%. During the course of time-lapse movies, the average fluorescence of invasive phage DNA in cells with CRISPR activity, decays more rapidly compared to cells without, and phage DNA is fully degraded by around 44 minutes on average. Moreover, the degradation appears to be independent of cell size or the phage DNA ejection site suggesting that Cas proteins are dispersed in sufficient quantities throughout the cell. With the CRISPR-Cas visualization system we developed, we are able to examine and characterize how a CRISPR system degrades invading phage DNA at the single-cell level. This work provides direct evidence and improves the current understanding on how CRISPR breaks down invading DNA.

Development of Nano/Micro Probes for Femtoliter Volume and Single Cell Measurements

NASA Astrophysics Data System (ADS)

Gao, Yang

Single cell analysis has recently emerged as an important field of biomedical re- search. It is now clear that heterogeneity of cell metabolism functions in complex biological systems is correlated to changes in biological function and disease processes. A variety of nano/micro probes were developed to enable investigation of cells properties such as membrane stiffness, pH value. However, very few designs were focused on single cell metabolic function studies. There is a critical need for technologies that provide analysis of heterogeneity of cell metabolic functions, especially on metabolism. Nevertheless, the few existing approaches suffer from fundamental defects and need to be improved. This work focused on developing nano/micro probes that are suitable for single cell functionality investigation. Both types of probes are designed to measure cell-to-cell/time-to-time heterogeneity in metabolic functions over a long period of time. Lab-made carbon nanoprobes were developed especially for electro-physiological measurement. The unique structure of the carbon nanoprobes makes them suitable for important intracellular applications like trans-membrane potential measurements and various electrochemical measurement for cell function studies. While it is important of have ability to carry out intracellular measure, there are also occasions where the information of a cell as a whole is collected. One of the most important indicator of a cells metabolic functions is cell respiration rate/oxygen consumption rate. A micro-perfusion based multi-functional single cell sensing probe was the developed to carry out measurements on cell as a whole. Formed by a double-barrel theta pipette, the perfusion flow enables the direct measurement of the metabolic flux for example oxygen consumption rate. In conclusion, this work developed nano/micro-probes as novel single cell investigation tools. The data acquired from these tools could provide valuable assistance on applications including cell metabolism studies, cancer diagnoses, and therapy evaluations.

Development of a facile droplet-based single-cell isolation platform for cultivation and genomic analysis in microorganisms.

PubMed

Zhang, Qiang; Wang, Tingting; Zhou, Qian; Zhang, Peng; Gong, Yanhai; Gou, Honglei; Xu, Jian; Ma, Bo

2017-01-23

Wider application of single-cell analysis has been limited by the lack of an easy-to-use and low-cost strategy for single-cell isolation that can be directly coupled to single-cell sequencing and single-cell cultivation, especially for small-size microbes. Herein, a facile droplet microfluidic platform was developed to dispense individual microbial cells into conventional standard containers for downstream analysis. Functional parts for cell encapsulation, droplet inspection and sorting, as well as a chip-to-tube capillary interface were integrated on one single chip with simple architecture, and control of the droplet sorting was achieved by a low-cost solenoid microvalve. Using microalgal and yeast cells as models, single-cell isolation success rate of over 90% and single-cell cultivation success rate of 80% were demonstrated. We further showed that the individual cells isolated can be used in high-quality DNA and RNA analyses at both gene-specific and whole-genome levels (i.e. real-time quantitative PCR and genome sequencing). The simplicity and reliability of the method should improve accessibility of single-cell analysis and facilitate its wider application in microbiology researches.

Development of a facile droplet-based single-cell isolation platform for cultivation and genomic analysis in microorganisms

PubMed Central

Zhang, Qiang; Wang, Tingting; Zhou, Qian; Zhang, Peng; Gong, Yanhai; Gou, Honglei; Xu, Jian; Ma, Bo

2017-01-01

Wider application of single-cell analysis has been limited by the lack of an easy-to-use and low-cost strategy for single-cell isolation that can be directly coupled to single-cell sequencing and single-cell cultivation, especially for small-size microbes. Herein, a facile droplet microfluidic platform was developed to dispense individual microbial cells into conventional standard containers for downstream analysis. Functional parts for cell encapsulation, droplet inspection and sorting, as well as a chip-to-tube capillary interface were integrated on one single chip with simple architecture, and control of the droplet sorting was achieved by a low-cost solenoid microvalve. Using microalgal and yeast cells as models, single-cell isolation success rate of over 90% and single-cell cultivation success rate of 80% were demonstrated. We further showed that the individual cells isolated can be used in high-quality DNA and RNA analyses at both gene-specific and whole-genome levels (i.e. real-time quantitative PCR and genome sequencing). The simplicity and reliability of the method should improve accessibility of single-cell analysis and facilitate its wider application in microbiology researches. PMID:28112223

Single cell digital polymerase chain reaction on self-priming compartmentalization chip

PubMed Central

Zhu, Qiangyuan; Qiu, Lin; Xu, Yanan; Li, Guang; Mu, Ying

2017-01-01

Single cell analysis provides a new framework for understanding biology and disease, however, an absolute quantification of single cell gene expression still faces many challenges. Microfluidic digital polymerase chain reaction (PCR) provides a unique method to absolutely quantify the single cell gene expression, but only limited devices are developed to analyze a single cell with detection variation. This paper describes a self-priming compartmentalization (SPC) microfluidic digital polymerase chain reaction chip being capable of performing single molecule amplification from single cell. The chip can be used to detect four single cells simultaneously with 85% of sample digitization. With the optimized protocol for the SPC chip, we first tested the ability, precision, and sensitivity of our SPC digital PCR chip by assessing β-actin DNA gene expression in 1, 10, 100, and 1000 cells. And the reproducibility of the SPC chip is evaluated by testing 18S rRNA of single cells with 1.6%–4.6% of coefficient of variation. At last, by detecting the lung cancer related genes, PLAU gene expression of A549 cells at the single cell level, the single cell heterogeneity was demonstrated. So, with the power-free, valve-free SPC chip, the gene copy number of single cells can be quantified absolutely with higher sensitivity, reduced labor time, and reagent. We expect that this chip will enable new studies for biology and disease. PMID:28191267

Single cell digital polymerase chain reaction on self-priming compartmentalization chip.

PubMed

Zhu, Qiangyuan; Qiu, Lin; Xu, Yanan; Li, Guang; Mu, Ying

2017-01-01

Single cell analysis provides a new framework for understanding biology and disease, however, an absolute quantification of single cell gene expression still faces many challenges. Microfluidic digital polymerase chain reaction (PCR) provides a unique method to absolutely quantify the single cell gene expression, but only limited devices are developed to analyze a single cell with detection variation. This paper describes a self-priming compartmentalization (SPC) microfluidic digital polymerase chain reaction chip being capable of performing single molecule amplification from single cell. The chip can be used to detect four single cells simultaneously with 85% of sample digitization. With the optimized protocol for the SPC chip, we first tested the ability, precision, and sensitivity of our SPC digital PCR chip by assessing β-actin DNA gene expression in 1, 10, 100, and 1000 cells. And the reproducibility of the SPC chip is evaluated by testing 18S rRNA of single cells with 1.6%-4.6% of coefficient of variation. At last, by detecting the lung cancer related genes, PLAU gene expression of A549 cells at the single cell level, the single cell heterogeneity was demonstrated. So, with the power-free, valve-free SPC chip, the gene copy number of single cells can be quantified absolutely with higher sensitivity, reduced labor time, and reagent. We expect that this chip will enable new studies for biology and disease.

Motion of single MreB bacterial actin proteins in Caulobacter show treadmilling in vivo

NASA Astrophysics Data System (ADS)

Moerner, W. E.; Kim, Soyeon; Gitai, Zemer; Kinkhabwala, Anika; McAdams, Harley; Shapiro, Lucy

2006-03-01

Ensemble imaging of a bacterial actin homologue, the MreB protein, suggests that the MreB proteins form a dynamic filamentous spiral along the long axis of the cell in Caulobacter crescentus. MreB contracts and expands along the cell axis and plays an important role in cell shape and polarity maintenance, as well as chromosome segregation and translocation of the origin of replication during cell division. In this study we investigated the real-time polymerization of MreB in Caulobacter crescentus using single-molecule fluorescence imaging. With time-lapse imaging, polymerized MreB could be distinguished from cytoplasmic MreB monomers, because single monomeric MreB showed fast motion characteristic of Brownian diffusion, while single polymerized MreB displayed slow, directed motion. This directional movement of labeled MreB in the growing polymer implies that treadmilling is the predominant mechanism in MreB filament formation. These single-molecule imaging experiments provide the first available information on the velocity of bacterial actin polymerization in a living cell.

Cancer cell classification with coherent diffraction imaging using an extreme ultraviolet radiation source

PubMed Central

Zürch, Michael; Foertsch, Stefan; Matzas, Mark; Pachmann, Katharina; Kuth, Rainer; Spielmann, Christian

2014-01-01

Abstract. In cancer treatment, it is highly desirable to classify single cancer cells in real time. The standard method is polymerase chain reaction requiring a substantial amount of resources and time. Here, we present an innovative approach for rapidly classifying different cell types: we measure the diffraction pattern of a single cell illuminated with coherent extreme ultraviolet (XUV) laser-generated radiation. These patterns allow distinguishing different breast cancer cell types in a subsequent step. Moreover, the morphology of the object can be retrieved from the diffraction pattern with submicron resolution. In a proof-of-principle experiment, we prepared single MCF7 and SKBR3 breast cancer cells on gold-coated silica slides. The output of a laser-driven XUV light source is focused onto a single unstained and unlabeled cancer cell. With the resulting diffraction pattern, we could clearly identify the different cell types. With an improved setup, it will not only be feasible to classify circulating tumor cells with a high throughput, but also to identify smaller objects such as bacteria or even viruses. PMID:26158049

Microfluidic guillotine for single-cell wound repair studies

NASA Astrophysics Data System (ADS)

Blauch, Lucas R.; Gai, Ya; Khor, Jian Wei; Sood, Pranidhi; Marshall, Wallace F.; Tang, Sindy K. Y.

2017-07-01

Wound repair is a key feature distinguishing living from nonliving matter. Single cells are increasingly recognized to be capable of healing wounds. The lack of reproducible, high-throughput wounding methods has hindered single-cell wound repair studies. This work describes a microfluidic guillotine for bisecting single Stentor coeruleus cells in a continuous-flow manner. Stentor is used as a model due to its robust repair capacity and the ability to perform gene knockdown in a high-throughput manner. Local cutting dynamics reveals two regimes under which cells are bisected, one at low viscous stress where cells are cut with small membrane ruptures and high viability and one at high viscous stress where cells are cut with extended membrane ruptures and decreased viability. A cutting throughput up to 64 cells per minute—more than 200 times faster than current methods—is achieved. The method allows the generation of more than 100 cells in a synchronized stage of their repair process. This capacity, combined with high-throughput gene knockdown in Stentor, enables time-course mechanistic studies impossible with current wounding methods.

Cancer cell classification with coherent diffraction imaging using an extreme ultraviolet radiation source.

PubMed

Zürch, Michael; Foertsch, Stefan; Matzas, Mark; Pachmann, Katharina; Kuth, Rainer; Spielmann, Christian

2014-10-01

In cancer treatment, it is highly desirable to classify single cancer cells in real time. The standard method is polymerase chain reaction requiring a substantial amount of resources and time. Here, we present an innovative approach for rapidly classifying different cell types: we measure the diffraction pattern of a single cell illuminated with coherent extreme ultraviolet (XUV) laser-generated radiation. These patterns allow distinguishing different breast cancer cell types in a subsequent step. Moreover, the morphology of the object can be retrieved from the diffraction pattern with submicron resolution. In a proof-of-principle experiment, we prepared single MCF7 and SKBR3 breast cancer cells on gold-coated silica slides. The output of a laser-driven XUV light source is focused onto a single unstained and unlabeled cancer cell. With the resulting diffraction pattern, we could clearly identify the different cell types. With an improved setup, it will not only be feasible to classify circulating tumor cells with a high throughput, but also to identify smaller objects such as bacteria or even viruses.

Single-cell intracellular nano-pH probes.

PubMed

Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

2015-01-01

Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution.

Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells

PubMed Central

Eroglu, Emrah; Rost, Rene; Bischof, Helmut; Blass, Sandra; Schreilechner, Anna; Gottschalk, Benjamin; Depaoli, Maria R.; Klec, Christiane; Charoensin, Suphachai; Madreiter-Sokolowski, Corina T.; Ramadani, Jeta; Waldeck-Weiermair, Markus; Graier, Wolfgang F.; Malli, Roland

2017-01-01

Nitric Oxide (NO•) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO• in vivo and in vitro, the real-time monitoring of NO• at the single-cell level is very challenging. The physiological or pathological effects of NO• are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO• are highly desirable. Recently, we expanded the pallet of NO• indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO•) probes (geNOps) that directly respond to cellular NO• fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO• signals in response to two different chemical NO•-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO• levels as compared with the inorganic NO• donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO• formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO• signals in diverse experimental setups. PMID:28362417

Two-dimensional single-cell patterning with one cell per well driven by surface acoustic waves

PubMed Central

Collins, David J.; Morahan, Belinda; Garcia-Bustos, Jose; Doerig, Christian; Plebanski, Magdalena; Neild, Adrian

2015-01-01

In single-cell analysis, cellular activity and parameters are assayed on an individual, rather than population-average basis. Essential to observing the activity of these cells over time is the ability to trap, pattern and retain them, for which previous single-cell-patterning work has principally made use of mechanical methods. While successful as a long-term cell-patterning strategy, these devices remain essentially single use. Here we introduce a new method for the patterning of multiple spatially separated single particles and cells using high-frequency acoustic fields with one cell per acoustic well. We characterize and demonstrate patterning for both a range of particle sizes and the capture and patterning of cells, including human lymphocytes and red blood cells infected by the malarial parasite Plasmodium falciparum. This ability is made possible by a hitherto unexplored regime where the acoustic wavelength is on the same order as the cell dimensions. PMID:26522429

Limiting Energy Dissipation Induces Glassy Kinetics in Single-Cell High-Precision Responses

PubMed Central

Das, Jayajit

2016-01-01

Single cells often generate precise responses by involving dissipative out-of-thermodynamic-equilibrium processes in signaling networks. The available free energy to fuel these processes could become limited depending on the metabolic state of an individual cell. How does limiting dissipation affect the kinetics of high-precision responses in single cells? I address this question in the context of a kinetic proofreading scheme used in a simple model of early-time T cell signaling. Using exact analytical calculations and numerical simulations, I show that limiting dissipation qualitatively changes the kinetics in single cells marked by emergence of slow kinetics, large cell-to-cell variations of copy numbers, temporally correlated stochastic events (dynamic facilitation), and ergodicity breaking. Thus, constraints in energy dissipation, in addition to negatively affecting ligand discrimination in T cells, can create a fundamental difficulty in determining single-cell kinetics from cell-population results. PMID:26958894

In vitro culture of individual mouse preimplantation embryos: the role of embryo density, microwells, oxygen, timing and conditioned media.

PubMed

Kelley, Rebecca L; Gardner, David K

2017-05-01

Single embryo culture is suboptimal compared with group culture, but necessary for embryo monitoring, and culture systems should be improved for single embryos. Pronucleate mouse embryos were used to assess the effect of culture conditions on single embryo development. Single culture either before or after compaction reduced cell numbers (112.2 ± 3.1; 110.2 ± 3.5) compared with group culture throughout (127.0 ± 3.4; P < 0.05). Reduction of media volume from 20 µl to 2 µl increased blastocyst cell numbers in single embryos cultured in 5% oxygen (84.4 ± 3.2 versus 97.8 ± 2.8; P < 0.05), but not in 20% oxygen (55.2 ± 2.9 versus 57.1 ± 2.8). Culture in microwell plates for the EmbryoScope and Primo Vision time-lapse systems changed cleavage timings and increased inner cell mass cell number (24.1 ± 1.0; 23.4 ± 1.2) compared with a 2 µl microdrop (18.4 ± 1.0; P < 0.05). Addition of embryo-conditioned media to single embryos increased hatching rate and blastocyst cell number (91.5 ± 4.7 versus 113.1 ± 4.4; P < 0.01). Single culture before or after compaction is therefore detrimental; oxygen, media volume and microwells influence single embryo development; and embryo-conditioned media may substitute for group culture. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Single Cell and Metagenomic Assemblies: Biology Drives Technical Choices and Goals (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

ScienceCinema

Stepanauskas, Ramunas

2018-02-06

DOE JGI's Tanja Woyke, chair of the Single Cells and Metagenomes session, delivers an introduction, followed by Bigelow Laboratory's Ramunas Stepanauskas on "Single Cell and Metagenomic Assemblies: Biology Drives Technical Choices and Goals" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

Single chamber microbial fuel cell with Ni-Co cathode

NASA Astrophysics Data System (ADS)

Włodarczyk, Barbara; Włodarczyk, Paweł P.; Kalinichenko, Antonina

2017-10-01

The possibility of wastewater treatment and the parallel energy production using the Ni-Co alloy as cathode catalyst for single chamber microbial fuel cells is presented in this research. The research included a preparation of catalyst and comparison of COD, NH4+ and NO3- reduction in the reactor without aeration, with aeration and with using a single chamber microbial fuel cell with Ni-Co cathode. The reduction time for COD with the use of microbial fuel cell with the Ni-Co catalyst is similar to the reduction time with aeration. The current density (2.4 A·m-2) and amount of energy (0.48 Wh) obtained in MFC is low, but the obtained amount of energy allows elimination of the energy needed for reactor aeration. It has been shown that the Ni-Co can be used as cathode catalyst in single chamber microbial fuel cells.

Deconstructing stem cell population heterogeneity: Single-cell analysis and modeling approaches

PubMed Central

Wu, Jincheng; Tzanakakis, Emmanuel S.

2014-01-01

Isogenic stem cell populations display cell-to-cell variations in a multitude of attributes including gene or protein expression, epigenetic state, morphology, proliferation and proclivity for differentiation. The origins of the observed heterogeneity and its roles in the maintenance of pluripotency and the lineage specification of stem cells remain unclear. Addressing pertinent questions will require the employment of single-cell analysis methods as traditional cell biochemical and biomolecular assays yield mostly population-average data. In addition to time-lapse microscopy and flow cytometry, recent advances in single-cell genomic, transcriptomic and proteomic profiling are reviewed. The application of multiple displacement amplification, next generation sequencing, mass cytometry and spectrometry to stem cell systems is expected to provide a wealth of information affording unprecedented levels of multiparametric characterization of cell ensembles under defined conditions promoting pluripotency or commitment. Establishing connections between single-cell analysis information and the observed phenotypes will also require suitable mathematical models. Stem cell self-renewal and differentiation are orchestrated by the coordinated regulation of subcellular, intercellular and niche-wide processes spanning multiple time scales. Here, we discuss different modeling approaches and challenges arising from their application to stem cell populations. Integrating single-cell analysis with computational methods will fill gaps in our knowledge about the functions of heterogeneity in stem cell physiology. This combination will also aid the rational design of efficient differentiation and reprogramming strategies as well as bioprocesses for the production of clinically valuable stem cell derivatives. PMID:24035899

Dynamic generation of concentration- and temporal-dependent chemical signals in an integrated microfluidic device for single-cell analysis.

PubMed

Gonzalez-Suarez, Alan Mauricio; Peña-Del Castillo, Johanna G; Hernandez-Cruz, Arturo; Garcia-Cordero, Jose Luis

2018-06-19

Intracellular signaling pathways are affected by the temporal nature of external chemical signaling molecules such as neuro-transmitters or hormones. Developing high-throughput technologies to mimic these time-varying chemical signals and to analyze the response of single cells would deepen our understanding of signaling networks. In this work, we introduce a microfluidic platform to stimulate hundreds of single cells with chemical waveforms of tunable frequency and amplitude. Our device produces a linear gradient of 9 concentrations that are delivered to an equal number of chambers, each containing 492 microwells, where individual cells are captured. The device can alternate between the different stimuli concentrations and a control buffer, with a maximum operating frequency of 33 mHz that can be adjusted from a computer. Fluorescent time-lapse microscopy enables to obtain hundreds of thousands of data points from one experiment. We characterized the gradient performance and stability by staining hundreds of cells with calcein AM. We also assessed the capacity of our device to introduce periodic chemical stimuli of different amplitudes and frequencies. To demonstrate our device performance, we studied the dynamics of intracellular Ca2+ release from intracellular stores of HEK cells when stimulated with carbachol at 4.5 and 20 mHz. Our work opens the possibility of characterizing the dynamic responses in real time of signaling molecules to time-varying chemical stimuli with single cell resolution.

Characteristics of single Ca(2+) channel kinetics in feline hypertrophied ventricular myocytes.

PubMed

Yang, Xiangjun; Hui, Jie; Jiang, Tingbo; Song, Jianping; Liu, Zhihua; Jiang, Wenping

2002-04-01

To explore the mechanism underlying the prolongation of action potential and delayed inactivation of the L-type Ca(2+) (I(Ca, L)) current in a feline model of left ventricular system hypertension and concomitant hypertrophy. Single Ca(2+) channel properties in myocytes isolated from normal and pressure overloaded cat left ventricles were studied, using patch-clamp techniques. Left ventricular pressure overload was induced by partial ligation of the ascending aorta for 4 - 6 weeks. The amplitude of single Ca(2+) channel current evoked by depolarizing pulses from -40 mV to 0 mV was 1.02 +/- 0.03 pA in normal cells and 1.05 +/- 0.03 pA in hypertrophied cells, and there was no difference in single channel current-voltage relationships between the groups since slope conductance was 26.2 +/- 1.0 pS in normal and hypertrophied cells, respectively. Peak amplitudes of the ensemble-averaged single Ca(2+) channel currents were not different between the two groups of cells. However, the amplitude of this averaged current at the end of the clamp pulse was significantly larger in hypertrophied cells than in normal cells. Open-time histograms revealed that open-time distribution was fitted by a single exponential function in channels of normal cells and by a two exponential function in channels of hypertrophied cells. The number of long-lasting openings was increased in channels of hypertrophied cells, and therefore the calculated mean open time of the channel was significantly longer compared to normal controls. Kinetic changes in the Ca(2+) channel may underlie both hypertrophy-associated delayed inactivation of the Ca(2+) current and, in part, the pressure overload-induced action potential lengthening in this cat model of ventricular left systolic hypertension and hypertrophy.

A disposable picolitre bioreactor for cultivation and investigation of industrially relevant bacteria on the single cell level.

PubMed

Grünberger, Alexander; Paczia, Nicole; Probst, Christopher; Schendzielorz, Georg; Eggeling, Lothar; Noack, Stephan; Wiechert, Wolfgang; Kohlheyer, Dietrich

2012-05-08

In the continuously growing field of industrial biotechnology the scale-up from lab to industrial scale is still a major hurdle to develop competitive bioprocesses. During scale-up the productivity of single cells might be affected by bioreactor inhomogeneity and population heterogeneity. Currently, these complex interactions are difficult to investigate. In this report, design, fabrication and operation of a disposable picolitre cultivation system is described, in which environmental conditions can be well controlled on a short time scale and bacterial microcolony growth experiments can be observed by time-lapse microscopy. Three exemplary investigations will be discussed emphasizing the applicability and versatility of the device. Growth and analysis of industrially relevant bacteria with single cell resolution (in particular Escherichia coli and Corynebacterium glutamicum) starting from one single mother cell to densely packed cultures is demonstrated. Applying the picolitre bioreactor, 1.5-fold increased growth rates of C. glutamicum wild type cells were observed compared to typical 1 litre lab-scale batch cultivation. Moreover, the device was used to analyse and quantify the morphological changes of an industrially relevant l-lysine producer C. glutamicum after artificially inducing starvation conditions. Instead of a one week lab-scale experiment, only 1 h was sufficient to reveal the same information. Furthermore, time lapse microscopy during 24 h picolitre cultivation of an arginine producing strain containing a genetically encoded fluorescence sensor disclosed time dependent single cell productivity and growth, which was not possible with conventional methods.

Imaging burst kinetics and spatial coordination during serial killing by single natural killer cells

PubMed Central

Choi, Paul J.; Mitchison, Timothy J.

2013-01-01

Cytotoxic lymphocytes eliminate virus-infected and cancerous cells by immune recognition and killing through the perforin-granzyme pathway. Traditional killing assays measure average target cell lysis at fixed times and high effector:target ratios. Such assays obscure kinetic details that might reveal novel physiology. We engineered target cells to report on granzyme activity, used very low effector:target ratios to observe potential serial killing, and performed low magnification time-lapse imaging to reveal time-dependent statistics of natural killer (NK) killing at the single-cell level. Most kills occurred during serial killing, and a single NK cell killed up to 10 targets over a 6-h assay. The first kill was slower than subsequent kills, especially on poor targets, or when NK signaling pathways were partially inhibited. Spatial analysis showed that sequential kills were usually adjacent. We propose that NK cells integrate signals from the previous and current target, possibly by simultaneous contact. The resulting burst kinetics and spatial coordination may control the activity of NK cells in tissues. PMID:23576740

Evaluation of the Cow Rumen Metagenome: Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

ScienceCinema

Sczyrba, Alex

2018-02-13

DOE JGI's Alex Sczyrba on "Evaluation of the Cow Rumen Metagenome" and "Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

Evaluation of the Cow Rumen Metagenome: Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sczyrba, Alex

2011-10-13

DOE JGI's Alex Sczyrba on "Evaluation of the Cow Rumen Metagenome" and "Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

On-chip manipulation of single microparticles, cells, and organisms using surface acoustic waves.

PubMed

Ding, Xiaoyun; Lin, Sz-Chin Steven; Kiraly, Brian; Yue, Hongjun; Li, Sixing; Chiang, I-Kao; Shi, Jinjie; Benkovic, Stephen J; Huang, Tony Jun

2012-07-10

Techniques that can dexterously manipulate single particles, cells, and organisms are invaluable for many applications in biology, chemistry, engineering, and physics. Here, we demonstrate standing surface acoustic wave based "acoustic tweezers" that can trap and manipulate single microparticles, cells, and entire organisms (i.e., Caenorhabditis elegans) in a single-layer microfluidic chip. Our acoustic tweezers utilize the wide resonance band of chirped interdigital transducers to achieve real-time control of a standing surface acoustic wave field, which enables flexible manipulation of most known microparticles. The power density required by our acoustic device is significantly lower than its optical counterparts (10,000,000 times less than optical tweezers and 100 times less than optoelectronic tweezers), which renders the technique more biocompatible and amenable to miniaturization. Cell-viability tests were conducted to verify the tweezers' compatibility with biological objects. With its advantages in biocompatibility, miniaturization, and versatility, the acoustic tweezers presented here will become a powerful tool for many disciplines of science and engineering.

Image segmentation and dynamic lineage analysis in single-cell fluorescence microscopy.

PubMed

Wang, Quanli; Niemi, Jarad; Tan, Chee-Meng; You, Lingchong; West, Mike

2010-01-01

An increasingly common component of studies in synthetic and systems biology is analysis of dynamics of gene expression at the single-cell level, a context that is heavily dependent on the use of time-lapse movies. Extracting quantitative data on the single-cell temporal dynamics from such movies remains a major challenge. Here, we describe novel methods for automating key steps in the analysis of single-cell, fluorescent images-segmentation and lineage reconstruction-to recognize and track individual cells over time. The automated analysis iteratively combines a set of extended morphological methods for segmentation, and uses a neighborhood-based scoring method for frame-to-frame lineage linking. Our studies with bacteria, budding yeast and human cells, demonstrate the portability and usability of these methods, whether using phase, bright field or fluorescent images. These examples also demonstrate the utility of our integrated approach in facilitating analyses of engineered and natural cellular networks in diverse settings. The automated methods are implemented in freely available, open-source software.

Real-time detection and monitoring of the drug resistance of single myeloid leukemia cells by diffused total internal reflection.

PubMed

Liang, L; Jin, Y X; Zhu, X Q; Zhou, F L; Yang, Y

2018-05-15

Real-time detection and monitoring of the drug resistance of single cells have important significance in clinical diagnosis and therapy. Traditional methods operate a number of times for each individual concentration, and innovation is required for the design of more simple and efficient manipulation platforms with necessary higher sensitivity. Here, we have developed a novel diffused total internal reflection (TIR) method to perform drug metabolism and cytotoxicity analysis of trapped myeloid leukemia cells. Molm-13 cells, a type of acute myeloid leukemia cell, were chosen and injected into the device and fittingly captured by cell traps. Differing from previous studies, a series of different concentrations of azelaic acid (AZA) drug could be used from 0 mM to 50 mM through convection and diffusion processes in a single chip, with each concentration region featuring 50 cells, with a total of 549 cell trapping units. Thanks to the high sensitivity of the TIR method, only cells with the same drug concentration could be illuminated in the detection process. By adjusting the incident angle, we could exactly detect and monitor the drug resistance of the cells using different drug concentrations and the experimental resolution of the drug concentration was as small as 5 mM. Images of the membrane integrity and morphology of the cells in the bright field were measured and we also monitored the cell viabilities in the dark field over 2 hours. The effects of AZA on the Molm-13 cells were explored in different concentrations at the single cell level. Compared with the results of the traditional MTT assay method, the experimental results are more simple and accurate. A cell death of 5% at an AZA concentration of 5 mM was observed after 30 minutes, while a concentration of 40 mM corresponded to a 98% cell death. The designed method in this study provides a novel toolkit to control and monitor drug resistance at the single cell level more easily with higher sensitivity and we believe it has significant potential application in single cell quality assessment and medicine analysis in clinical practice.

Integrated time-lapse and single-cell transcription studies highlight the variable and dynamic nature of human hematopoietic cell fate commitment

PubMed Central

Moussy, Alice; Cosette, Jérémie; Parmentier, Romuald; da Silva, Cindy; Corre, Guillaume; Richard, Angélique; Gandrillon, Olivier; Stockholm, Daniel

2017-01-01

Individual cells take lineage commitment decisions in a way that is not necessarily uniform. We address this issue by characterising transcriptional changes in cord blood-derived CD34+ cells at the single-cell level and integrating data with cell division history and morphological changes determined by time-lapse microscopy. We show that major transcriptional changes leading to a multilineage-primed gene expression state occur very rapidly during the first cell cycle. One of the 2 stable lineage-primed patterns emerges gradually in each cell with variable timing. Some cells reach a stable morphology and molecular phenotype by the end of the first cell cycle and transmit it clonally. Others fluctuate between the 2 phenotypes over several cell cycles. Our analysis highlights the dynamic nature and variable timing of cell fate commitment in hematopoietic cells, links the gene expression pattern to cell morphology, and identifies a new category of cells with fluctuating phenotypic characteristics, demonstrating the complexity of the fate decision process (which is different from a simple binary switch between 2 options, as it is usually envisioned). PMID:28749943

Real-time imaging of specific genomic loci in eukaryotic cells using the ANCHOR DNA labelling system.

PubMed

Germier, Thomas; Sylvain, Audibert; Silvia, Kocanova; David, Lane; Kerstin, Bystricky

2018-06-01

Spatio-temporal organization of the cell nucleus adapts to and regulates genomic processes. Microscopy approaches that enable direct monitoring of specific chromatin sites in single cells and in real time are needed to better understand the dynamics involved. In this chapter, we describe the principle and development of ANCHOR, a novel tool for DNA labelling in eukaryotic cells. Protocols for use of ANCHOR to visualize a single genomic locus in eukaryotic cells are presented. We describe an approach for live cell imaging of a DNA locus during the entire cell cycle in human breast cancer cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Single-cell intracellular nano-pH probes†

PubMed Central

Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

2016-01-01

Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution. PMID:27708772

Precise mass determination of single cell with cantilever-based microbiosensor system.

PubMed

Łabędź, Bogdan; Wańczyk, Aleksandra; Rajfur, Zenon

2017-01-01

Having determined the mass of a single cell of brewer yeast Saccharomyces cerevisiae by means of a microcantilever-based biosensor Cantisens CSR-801 (Concentris, Basel, Switzerland), it was found that its dry mass is 47,65 ± 1,05 pg. Found to be crucial in this mass determination was the cell position along the length of the cantilever. Moreover, calculations including cells positions on the cantilever provide a threefold better degree of accuracy than those which assume uniform mass distribution. We have also examined the influence of storage time on the single cell mass. Our results show that after 6 months there is an increase in the average mass of a single yeast cell.

Electrokinetic gated injection-based microfluidic system for quantitative analysis of hydrogen peroxide in individual HepG2 cells.

PubMed

Zhang, Xinyuan; Li, Qingling; Chen, Zhenzhen; Li, Hongmin; Xu, Kehua; Zhang, Lisheng; Tang, Bo

2011-03-21

A microfluidic system to determine hydrogen peroxide (H(2)O(2)) in individual HepG2 cells based on the electrokinetic gated injection was developed for the first time. A home-synthesized fluorescent probe, bis(p-methylbenzenesulfonate)dichlorofluorescein (FS), was employed to label intracellular H(2)O(2) in the intact cells. On a simple cross microchip, multiple single-cell operations, including single cell injection, cytolysis, electrophoresis separation and detection of H(2)O(2), were automatically carried out within 60 s using the electrokinetic gated injection and laser-induced fluorescence detection (LIFD). The performance of the method was evaluated under the optimal conditions. The linear calibration curve was over a range of 4.39-610 amol (R(2)=0.9994). The detection limit was 0.55 amol or 9.0×10(-10) M (S/N=3). The relative standard deviations (RSDs, n=6) of migration time and peak area were 1.4% and 4.8%, respectively. With the use of this method, the average content of H(2)O(2) in single HepG2 cells was found to be 16.09±9.84 amol (n=15). Separation efficiencies in excess of 17,000 theoretical plates for the cells were achieved. These results demonstrated that the efficient integration and automation of these single-cell operations enabled the sensitive, reproducible, and quantitative examination of intracellular H(2)O(2) at single-cell level. Owing to the advantages of simple microchip structure, controllable single-cell manipulation and ease in building, this platform provides a universal way to automatically determine other intracellular constituents within single cells. This journal is © The Royal Society of Chemistry 2011

A Single-Cell Biochemistry Approach Reveals PAR Complex Dynamics during Cell Polarization.

PubMed

Dickinson, Daniel J; Schwager, Francoise; Pintard, Lionel; Gotta, Monica; Goldstein, Bob

2017-08-21

Regulated protein-protein interactions are critical for cell signaling, differentiation, and development. For the study of dynamic regulation of protein interactions in vivo, there is a need for techniques that can yield time-resolved information and probe multiple protein binding partners simultaneously, using small amounts of starting material. Here we describe a single-cell protein interaction assay. Single-cell lysates are generated at defined time points and analyzed using single-molecule pull-down, yielding information about dynamic protein complex regulation in vivo. We established the utility of this approach by studying PAR polarity proteins, which mediate polarization of many animal cell types. We uncovered striking regulation of PAR complex composition and stoichiometry during Caenorhabditis elegans zygote polarization, which takes place in less than 20 min. PAR complex dynamics are linked to the cell cycle by Polo-like kinase 1 and govern the movement of PAR proteins to establish polarity. Our results demonstrate an approach to study dynamic biochemical events in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

Marker-free detection of progenitor cell differentiation by analysis of Brownian motion in micro-wells.

PubMed

Sekhavati, Farzad; Endele, Max; Rappl, Susanne; Marel, Anna-Kristina; Schroeder, Timm; Rädler, Joachim O

2015-02-01

The kinetics of stem and progenitor cell differentiation at the single-cell level provides essential clues to the complexity of the underlying decision-making circuits. In many hematopoietic progenitor cells, differentiation is accompanied by the expression of lineage-specific markers and by a transition from a non-adherent to an adherent state. Here, using the granulocyte-macrophage progenitor (GMP) as a model, we introduce a label-free approach that allows one to follow the course of this transition in hundreds of single cells in parallel. We trap single cells in patterned arrays of micro-wells and use phase-contrast time-lapse movies to distinguish non-adherent from adherent cells by an analysis of Brownian motion. This approach allowed us to observe the kinetics of induced differentiation of primary bone-marrow-derived GMPs into macrophages. The time lapse started 2 hours after addition of the cytokine M-CSF, and nearly 80% of the population had accomplished the transition within the first 20 h. The analysis of Brownian motion proved to be a sensitive and robust tool for monitoring the transition, and thus provides a high-throughput method for the study of cell differentiation at the single-cell level.

Limiting Energy Dissipation Induces Glassy Kinetics in Single-Cell High-Precision Responses.

PubMed

Das, Jayajit

2016-03-08

Single cells often generate precise responses by involving dissipative out-of-thermodynamic-equilibrium processes in signaling networks. The available free energy to fuel these processes could become limited depending on the metabolic state of an individual cell. How does limiting dissipation affect the kinetics of high-precision responses in single cells? I address this question in the context of a kinetic proofreading scheme used in a simple model of early-time T cell signaling. Using exact analytical calculations and numerical simulations, I show that limiting dissipation qualitatively changes the kinetics in single cells marked by emergence of slow kinetics, large cell-to-cell variations of copy numbers, temporally correlated stochastic events (dynamic facilitation), and ergodicity breaking. Thus, constraints in energy dissipation, in addition to negatively affecting ligand discrimination in T cells, can create a fundamental difficulty in determining single-cell kinetics from cell-population results. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Single-Cell Microfluidics to Study the Effects of Genome Deletion on Bacterial Growth Behavior.

PubMed

Yuan, Xiaofei; Couto, Jillian M; Glidle, Andrew; Song, Yanqing; Sloan, William; Yin, Huabing

2017-12-15

By directly monitoring single cell growth in a microfluidic platform, we interrogated genome-deletion effects in Escherichia coli strains. We compared the growth dynamics of a wild type strain with a clean genome strain, and their derived mutants at the single-cell level. A decreased average growth rate and extended average lag time were found for the clean genome strain, compared to those of the wild type strain. Direct correlation between the growth rate and lag time of individual cells showed that the clean genome population was more heterogeneous. Cell culturability (the ratio of growing cells to the sum of growing and nongrowing cells) of the clean genome population was also lower. Interestingly, after the random mutations induced by a glucose starvation treatment, for the clean genome population mutants that had survived the competition of chemostat culture, each parameter markedly improved (i.e., the average growth rate and cell culturability increased, and the lag time and heterogeneity decreased). However, this effect was not seen in the wild type strain; the wild type mutants cultured in a chemostat retained a high diversity of growth phenotypes. These results suggest that quasi-essential genes that were deleted in the clean genome might be required to retain a diversity of growth characteristics at the individual cell level under environmental stress. These observations highlight that single-cell microfluidics can reveal subtle individual cellular responses, enabling in-depth understanding of the population.

Single-cell-precision microplasma-induced cancer cell apoptosis.

PubMed

Tan, Xiao; Zhao, Shasha; Lei, Qian; Lu, Xinpei; He, Guangyuan; Ostrikov, Kostya

2014-01-01

The issue of single-cell control has recently attracted enormous interest. However, in spite of the presently achievable intracellular-level physiological probing through bio-photonics, nano-probe-based, and some other techniques, the issue of inducing selective, single-cell-precision apoptosis, without affecting neighbouring cells remains essentially open. Here we resolve this issue and report on the effective single-cell-precision cancer cell treatment using the reactive chemistry of the localized corona-type plasma discharge around a needle-like electrode with the spot size ∼1 µm. When the electrode is positioned with the micrometer precision against a selected cell, a focused and highly-localized micro-plasma discharge induces apoptosis in the selected individual HepG2 and HeLa cancer cells only, without affecting any surrounding cells, even in small cell clusters. This is confirmed by the real-time monitoring of the morphological and structural changes at the cellular and cell nucleus levels after the plasma exposure.

Electrical characterization of single cells using polysilicon wire ion sensor in an isolation window.

PubMed

Wu, You-Lin; Hsu, Po-Yen; Hsu, Chung-Ping; Wang, Chih-Cheng; Lee, Li-Wen; Lin, Jing-Jenn

2011-10-01

A polysilicon wire (PSW) sensor can detect the H(+) ion density (pH value) of the medium coated on its surface, and different cells produce different extracellular acidification and hence different H(+) ion densities. Based on this, we used a PSW sensor in combination with a mold-cast polydimethylsiloxane (PDMS) isolation window to detect the adhesion, apoptosis and extracellular acidification of single normal cells and single cancer cells. Single living human normal cells WI38, MRC5, and BEAS-2B as well as non-small-cell lung cancer (NSCLC) cells A549, H1299, and CH27 were cultivated separately inside the isolation window. The current flowing through the PSW channel was measured. From the PSW channel current change as a function of time, we determined the cell adhesion time by observing the time required for the current change to saturate, since a stable extracellular ion density was established after the cells were completely adhered to the PSW surface. The apoptosis of cells can also be determined when the channel current change drops to zero. We found that all the NSCLC cells had a higher channel current change and hence a lower pH value than the normal cells anytime after they were seeded. The corresponding average pH values were 5.86 for A549, 6.00 for H1299, 6.20 for CH27, 6.90 for BEAS-2B, 6.96for MRC5, and 7.02 for WI38, respectively, after the cells were completely adhered to the PSW surface. Our results show that NSCLC cells have a stronger cell-substrate adhesion and a higher extracellular acidification rate than normal cells.

Lung Injury; Relates to Real-Time Endoscopic Monitoring of Single Cells Respiratory Health in Lung

DTIC Science & Technology

2017-09-01

AWARD NUMBER: W81XWH-16-1-0253 TITLE: Lung Injury; Relates to Real- Time Endoscopic Monitoring of Single Cells Respiratory Health in Lung...and should not be construed as an official Department of the Army position, policy or decision unless so designated by other documentation. REPORT...response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and

Time-lapse electrical impedance spectroscopy for monitoring the cell cycle of single immobilized S. pombe cells.

PubMed

Zhu, Zhen; Frey, Olivier; Haandbaek, Niels; Franke, Felix; Rudolf, Fabian; Hierlemann, Andreas

2015-11-26

As a complement and alternative to optical methods, wide-band electrical impedance spectroscopy (EIS) enables multi-parameter, label-free and real-time detection of cellular and subcellular features. We report on a microfluidics-based system designed to reliably capture single rod-shaped Schizosaccharomyces pombe cells by applying suction through orifices in a channel wall. The system enables subsequent culturing of immobilized cells in an upright position, while dynamic changes in cell-cycle state and morphology were continuously monitored through EIS over a broad frequency range. Besides measuring cell growth, clear impedance signals for nuclear division have been obtained. The EIS system has been characterized with respect to sensitivity and detection limits. The spatial resolution in measuring cell length was 0.25 μm, which corresponds to approximately a 5-min interval of cell growth under standard conditions. The comprehensive impedance data sets were also used to determine the occurrence of nuclear division and cytokinesis. The obtained results have been validated through concurrent confocal imaging and plausibilized through comparison with finite-element modeling data. The possibility to monitor cellular and intracellular features of single S. pombe cells during the cell cycle at high spatiotemporal resolution renders the presented microfluidics-based EIS system a suitable tool for dynamic single-cell investigations.

Time-lapse electrical impedance spectroscopy for monitoring the cell cycle of single immobilized S. pombe cells

PubMed Central

Zhu, Zhen; Frey, Olivier; Haandbaek, Niels; Franke, Felix; Rudolf, Fabian; Hierlemann, Andreas

2015-01-01

As a complement and alternative to optical methods, wide-band electrical impedance spectroscopy (EIS) enables multi-parameter, label-free and real-time detection of cellular and subcellular features. We report on a microfluidics-based system designed to reliably capture single rod-shaped Schizosaccharomyces pombe cells by applying suction through orifices in a channel wall. The system enables subsequent culturing of immobilized cells in an upright position, while dynamic changes in cell-cycle state and morphology were continuously monitored through EIS over a broad frequency range. Besides measuring cell growth, clear impedance signals for nuclear division have been obtained. The EIS system has been characterized with respect to sensitivity and detection limits. The spatial resolution in measuring cell length was 0.25 μm, which corresponds to approximately a 5-min interval of cell growth under standard conditions. The comprehensive impedance data sets were also used to determine the occurrence of nuclear division and cytokinesis. The obtained results have been validated through concurrent confocal imaging and plausibilized through comparison with finite-element modeling data. The possibility to monitor cellular and intracellular features of single S. pombe cells during the cell cycle at high spatiotemporal resolution renders the presented microfluidics-based EIS system a suitable tool for dynamic single-cell investigations. PMID:26608589

One-Cell Doubling Evaluation by Living Arrays of Yeast, ODELAY!

DOE PAGES

Herricks, Thurston; Dilworth, David J.; Mast, Fred D.; ...

2016-11-16

Cell growth is a complex phenotype widely used in systems biology to gauge the impact of genetic and environmental perturbations. Due to the magnitude of genome-wide studies, resolution is often sacrificed in favor of throughput, creating a demand for scalable, time-resolved, quantitative methods of growth assessment. We present ODELAY (One-cell Doubling Evaluation by Living Arrays of Yeast), an automated and scalable growth analysis platform. High measurement density and single-cell resolution provide a powerful tool for large-scale multiparameter growth analysis based on the modeling of microcolony expansion on solid media. Pioneered in yeast but applicable to other colony forming organisms, ODELAYmore » extracts the three key growth parameters (lag time, doubling time, and carrying capacity) that define microcolony expansion from single cells, simultaneously permitting the assessment of population heterogeneity. The utility of ODELAY is illustrated using yeast mutants, revealing a spectrum of phenotypes arising from single and combinatorial growth parameter perturbations.« less

One-Cell Doubling Evaluation by Living Arrays of Yeast, ODELAY!

DOE Office of Scientific and Technical Information (OSTI.GOV)

Herricks, Thurston; Dilworth, David J.; Mast, Fred D.

Cell growth is a complex phenotype widely used in systems biology to gauge the impact of genetic and environmental perturbations. Due to the magnitude of genome-wide studies, resolution is often sacrificed in favor of throughput, creating a demand for scalable, time-resolved, quantitative methods of growth assessment. We present ODELAY (One-cell Doubling Evaluation by Living Arrays of Yeast), an automated and scalable growth analysis platform. High measurement density and single-cell resolution provide a powerful tool for large-scale multiparameter growth analysis based on the modeling of microcolony expansion on solid media. Pioneered in yeast but applicable to other colony forming organisms, ODELAYmore » extracts the three key growth parameters (lag time, doubling time, and carrying capacity) that define microcolony expansion from single cells, simultaneously permitting the assessment of population heterogeneity. The utility of ODELAY is illustrated using yeast mutants, revealing a spectrum of phenotypes arising from single and combinatorial growth parameter perturbations.« less

High-throughput, label-free, single-cell, microalgal lipid screening by machine-learning-equipped optofluidic time-stretch quantitative phase microscopy.

PubMed

Guo, Baoshan; Lei, Cheng; Kobayashi, Hirofumi; Ito, Takuro; Yalikun, Yaxiaer; Jiang, Yiyue; Tanaka, Yo; Ozeki, Yasuyuki; Goda, Keisuke

2017-05-01

The development of reliable, sustainable, and economical sources of alternative fuels to petroleum is required to tackle the global energy crisis. One such alternative is microalgal biofuel, which is expected to play a key role in reducing the detrimental effects of global warming as microalgae absorb atmospheric CO 2 via photosynthesis. Unfortunately, conventional analytical methods only provide population-averaged lipid amounts and fail to characterize a diverse population of microalgal cells with single-cell resolution in a non-invasive and interference-free manner. Here high-throughput label-free single-cell screening of lipid-producing microalgal cells with optofluidic time-stretch quantitative phase microscopy was demonstrated. In particular, Euglena gracilis, an attractive microalgal species that produces wax esters (suitable for biodiesel and aviation fuel after refinement), within lipid droplets was investigated. The optofluidic time-stretch quantitative phase microscope is based on an integration of a hydrodynamic-focusing microfluidic chip, an optical time-stretch quantitative phase microscope, and a digital image processor equipped with machine learning. As a result, it provides both the opacity and phase maps of every single cell at a high throughput of 10,000 cells/s, enabling accurate cell classification without the need for fluorescent staining. Specifically, the dataset was used to characterize heterogeneous populations of E. gracilis cells under two different culture conditions (nitrogen-sufficient and nitrogen-deficient) and achieve the cell classification with an error rate of only 2.15%. The method holds promise as an effective analytical tool for microalgae-based biofuel production. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Real-time Attack of LL-37 on Single Bacillus subtilis Cells

PubMed Central

Barns, Kenneth J.; Weisshaar, James C.

2013-01-01

Time-lapse fluorescence microscopy of single, growing Bacillus subtilis cells with 2-12 s time resolution reveals the mechanisms of antimicrobial peptide (AMP) action on a Gram-positive species with unprecedented detail. For the human cathelicidin LL-37 attacking B. subtilis, the symptoms of antimicrobial stress differ dramatically depending on the bulk AMP concentration. At 2 μM LL-37, the mean single-cell growth rate decreases, but membrane permeabilization does not occur. At 4 μM LL-37, cells abruptly shrink in size at the same time that Sytox Green enters the cytoplasm and stains the nucleoids. We interpret the shrinkage event as loss of turgor pressure (and presumably the membrane potential) due to permeabilization of the membrane. Movies of Sytox Green staining at 0.5 frame/s show that nucleoid staining is initially local, more consistent with pore formation than with global permeabilization models. In a novel “growth recovery” assay, cells are incubated with LL-37 for a variable period and then rinsed with fresh growth medium lacking LL-37. The growth rate attenuation observed at 2 μM LL-37 is a recoverable symptom, while the abrupt cell shrinkage observed at 4 μM LL-37 is not. PMID:23454084

Impact of jamming on collective cell migration

NASA Astrophysics Data System (ADS)

Nnetu, Kenechukwu David; Knorr, Melanie; Pawlizak, Steve; Fuhs, Thomas; Zink, Mareike; KäS, Josef A.

2012-02-01

Multi-cellular migration plays an important role in physiological processes such as embryogenesis, cancer metastasis and tissue repair. During migration, single cells undergo cycles of extension, adhesion and retraction resulting in morphological changes. In a confluent monolayer, there are inter-cellular interactions and crowding, however, the impact of these interactions on the dynamics and elasticity of the monolayer at the multi-cellular and single cell level is not well understood. Here we study the dynamics of a confluent epithelial monolayer by simultaneously measuring cell motion at the multi-cellular and single cell level for various cell densities and tensile elasticity. At the multi-cellular level, the system exhibited spatial kinetic transitions from isotropic to anisotropic migration on long times and the velocity of the monolayer decreased with increasing cell density. Moreover, the dynamics was spatially and temporally heterogeneous. Interestingly, the dynamics was also heterogeneous in wound-healing assays and the correlation length was fitted by compressed exponential. On the single cell scale, we observed transient caging effects with increasing cage rearrangement times as the system age due to an increase in density. Also, the density dependent elastic modulus of the monolayer scaled as a weak power law. Together, these findings suggest that caging effects at the single cell level initiates a slow and heterogeneous dynamics at the multi-cellular level which is similar to the glassy dynamics of deformable colloidal systems.

Visualization of IAV Genomes at the Single-Cell Level.

PubMed

Wang, Dan; Ma, Wenjun

2017-10-01

Different influenza A viruses (IAVs) infect the same cell in a host, and can subsequently produce new viruses through genome reassortment. By combining padlock probe RNA labeling with a single-cell analysis, a new approach effectively captures IAV genome trafficking and defines a time window for genome reassortment from same-cell coinfections. Copyright © 2017 Elsevier Ltd. All rights reserved.

Small is fast: astrocytic glucose and lactate metabolism at cellular resolution

PubMed Central

Barros, L. F.; San Martín, A.; Sotelo-Hitschfeld, T.; Lerchundi, R.; Fernández-Moncada, I.; Ruminot, I.; Gutiérrez, R.; Valdebenito, R.; Ceballo, S.; Alegría, K.; Baeza-Lehnert, F.; Espinoza, D.

2013-01-01

Brain tissue is highly dynamic in terms of electrical activity and energy demand. Relevant energy metabolites have turnover times ranging from milliseconds to seconds and are rapidly exchanged between cells and within cells. Until recently these fast metabolic events were inaccessible, because standard isotopic techniques require use of populations of cells and/or involve integration times of tens of minutes. Thanks to fluorescent probes and recently available genetically-encoded optical nanosensors, this Technology Report shows how it is now possible to monitor the concentration of metabolites in real-time and in single cells. In combination with ad hoc inhibitor-stop protocols, these probes have revealed a key role for K+ in the acute stimulation of astrocytic glycolysis by synaptic activity. They have also permitted detection of the Warburg effect in single cancer cells. Genetically-encoded nanosensors currently exist for glucose, lactate, NADH and ATP, and it is envisaged that other metabolite nanosensors will soon be available. These optical tools together with improved expression systems and in vivo imaging, herald an exciting era of single-cell metabolic analysis. PMID:23526722

Single-cell imaging techniques for the real-time detection of IP₃ in live cells.

PubMed

Nelson, Carl P

2013-01-01

Inositol 1,4,5-trisphosphate (IP(3)) is a ubiquitous second messenger, derived from the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP(2)) by enzymes of the phospholipase C (PLC) family. Binding of IP(3) to its cognate receptor in the endoplasmic reticulum membrane leads to release of Ca(2+) into the cytoplasm, which is involved in the regulation of an array of cellular functions. Traditional techniques for the detection of IP(3) have required the extraction of a large number of cells, with limitations in the time resolution of changes in IP(3) and an inability to obtain detailed information on the dynamics of this second messenger in single cells. Recent progress in this field has led to the development of a number of genetically encoded fluorescent biosensors, which upon recombinant expression are able selectively to detect real-time changes in IP(3) in single live cells. In this chapter, I detail protocols for the expression, visualization (by confocol or fluorescence microscopy), and interpretation of data obtained with such biosensors expressed in mammalian cells.

77 FR 21788 - Center for Scientific Review Notice of Closed Meetings

Federal Register 2010, 2011, 2012, 2013, 2014

2012-04-11

... Development, Stem Cell and Myocardial Regeneration. Date: May 10, 2012. Time: 2 p.m. to 3:30 p.m. Agenda: To... for Scientific Review Special Emphasis Panel; RFA Panel: Single Cell Analysis Reviews. Date: May 8... for Scientific Review Special Emphasis Panel; RFA Panel: Single Cell Analysis Reviews. Date: May 9...

Massively parallel digital transcriptional profiling of single cells

PubMed Central

Zheng, Grace X. Y.; Terry, Jessica M.; Belgrader, Phillip; Ryvkin, Paul; Bent, Zachary W.; Wilson, Ryan; Ziraldo, Solongo B.; Wheeler, Tobias D.; McDermott, Geoff P.; Zhu, Junjie; Gregory, Mark T.; Shuga, Joe; Montesclaros, Luz; Underwood, Jason G.; Masquelier, Donald A.; Nishimura, Stefanie Y.; Schnall-Levin, Michael; Wyatt, Paul W.; Hindson, Christopher M.; Bharadwaj, Rajiv; Wong, Alexander; Ness, Kevin D.; Beppu, Lan W.; Deeg, H. Joachim; McFarland, Christopher; Loeb, Keith R.; Valente, William J.; Ericson, Nolan G.; Stevens, Emily A.; Radich, Jerald P.; Mikkelsen, Tarjei S.; Hindson, Benjamin J.; Bielas, Jason H.

2017-01-01

Characterizing the transcriptome of individual cells is fundamental to understanding complex biological systems. We describe a droplet-based system that enables 3′ mRNA counting of tens of thousands of single cells per sample. Cell encapsulation, of up to 8 samples at a time, takes place in ∼6 min, with ∼50% cell capture efficiency. To demonstrate the system's technical performance, we collected transcriptome data from ∼250k single cells across 29 samples. We validated the sensitivity of the system and its ability to detect rare populations using cell lines and synthetic RNAs. We profiled 68k peripheral blood mononuclear cells to demonstrate the system's ability to characterize large immune populations. Finally, we used sequence variation in the transcriptome data to determine host and donor chimerism at single-cell resolution from bone marrow mononuclear cells isolated from transplant patients. PMID:28091601

Technologies for Single-Cell Isolation

PubMed Central

Gross, Andre; Schoendube, Jonas; Zimmermann, Stefan; Steeb, Maximilian; Zengerle, Roland; Koltay, Peter

2015-01-01

The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) respectively Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/dilution (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field. PMID:26213926

Technologies for Single-Cell Isolation.

PubMed

Gross, Andre; Schoendube, Jonas; Zimmermann, Stefan; Steeb, Maximilian; Zengerle, Roland; Koltay, Peter

2015-07-24

The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) respectively Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/dilution (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field.

SIDR: simultaneous isolation and parallel sequencing of genomic DNA and total RNA from single cells.

PubMed

Han, Kyung Yeon; Kim, Kyu-Tae; Joung, Je-Gun; Son, Dae-Soon; Kim, Yeon Jeong; Jo, Areum; Jeon, Hyo-Jeong; Moon, Hui-Sung; Yoo, Chang Eun; Chung, Woosung; Eum, Hye Hyeon; Kim, Sangmin; Kim, Hong Kwan; Lee, Jeong Eon; Ahn, Myung-Ju; Lee, Hae-Ock; Park, Donghyun; Park, Woong-Yang

2018-01-01

Simultaneous sequencing of the genome and transcriptome at the single-cell level is a powerful tool for characterizing genomic and transcriptomic variation and revealing correlative relationships. However, it remains technically challenging to analyze both the genome and transcriptome in the same cell. Here, we report a novel method for simultaneous isolation of genomic DNA and total RNA (SIDR) from single cells, achieving high recovery rates with minimal cross-contamination, as is crucial for accurate description and integration of the single-cell genome and transcriptome. For reliable and efficient separation of genomic DNA and total RNA from single cells, the method uses hypotonic lysis to preserve nuclear lamina integrity and subsequently captures the cell lysate using antibody-conjugated magnetic microbeads. Evaluating the performance of this method using real-time PCR demonstrated that it efficiently recovered genomic DNA and total RNA. Thorough data quality assessments showed that DNA and RNA simultaneously fractionated by the SIDR method were suitable for genome and transcriptome sequencing analysis at the single-cell level. The integration of single-cell genome and transcriptome sequencing by SIDR (SIDR-seq) showed that genetic alterations, such as copy-number and single-nucleotide variations, were more accurately captured by single-cell SIDR-seq compared with conventional single-cell RNA-seq, although copy-number variations positively correlated with the corresponding gene expression levels. These results suggest that SIDR-seq is potentially a powerful tool to reveal genetic heterogeneity and phenotypic information inferred from gene expression patterns at the single-cell level. © 2018 Han et al.; Published by Cold Spring Harbor Laboratory Press.

SIDR: simultaneous isolation and parallel sequencing of genomic DNA and total RNA from single cells

PubMed Central

Han, Kyung Yeon; Kim, Kyu-Tae; Joung, Je-Gun; Son, Dae-Soon; Kim, Yeon Jeong; Jo, Areum; Jeon, Hyo-Jeong; Moon, Hui-Sung; Yoo, Chang Eun; Chung, Woosung; Eum, Hye Hyeon; Kim, Sangmin; Kim, Hong Kwan; Lee, Jeong Eon; Ahn, Myung-Ju; Lee, Hae-Ock; Park, Donghyun; Park, Woong-Yang

2018-01-01

Simultaneous sequencing of the genome and transcriptome at the single-cell level is a powerful tool for characterizing genomic and transcriptomic variation and revealing correlative relationships. However, it remains technically challenging to analyze both the genome and transcriptome in the same cell. Here, we report a novel method for simultaneous isolation of genomic DNA and total RNA (SIDR) from single cells, achieving high recovery rates with minimal cross-contamination, as is crucial for accurate description and integration of the single-cell genome and transcriptome. For reliable and efficient separation of genomic DNA and total RNA from single cells, the method uses hypotonic lysis to preserve nuclear lamina integrity and subsequently captures the cell lysate using antibody-conjugated magnetic microbeads. Evaluating the performance of this method using real-time PCR demonstrated that it efficiently recovered genomic DNA and total RNA. Thorough data quality assessments showed that DNA and RNA simultaneously fractionated by the SIDR method were suitable for genome and transcriptome sequencing analysis at the single-cell level. The integration of single-cell genome and transcriptome sequencing by SIDR (SIDR-seq) showed that genetic alterations, such as copy-number and single-nucleotide variations, were more accurately captured by single-cell SIDR-seq compared with conventional single-cell RNA-seq, although copy-number variations positively correlated with the corresponding gene expression levels. These results suggest that SIDR-seq is potentially a powerful tool to reveal genetic heterogeneity and phenotypic information inferred from gene expression patterns at the single-cell level. PMID:29208629

Massively parallel single-molecule and single-cell emulsion reverse transcription polymerase chain reaction using agarose droplet microfluidics.

PubMed

Zhang, Huifa; Jenkins, Gareth; Zou, Yuan; Zhu, Zhi; Yang, Chaoyong James

2012-04-17

A microfluidic device for performing single copy, emulsion Reverse Transcription Polymerase Chain Reaction (RT-PCR) within agarose droplets is presented. A two-aqueous-inlet emulsion droplet generator was designed and fabricated to produce highly uniform monodisperse picoliter agarose emulsion droplets with RT-PCR reagents in carrier oil. Template RNA or cells were delivered from one inlet with RT-PCR reagents/cell lysis buffer delivered separately from the other. Efficient RNA/cell encapsulation and RT-PCR at the single copy level was achieved in agarose-in-oil droplets, which, after amplification, can be solidified into agarose beads for further analysis. A simple and efficient method to graft primer to the polymer matrix using 5'-acrydite primer was developed to ensure highly efficient trapping of RT-PCR products in agarose. High-throughput single RNA molecule/cell RT-PCR was demonstrated in stochastically diluted solutions. Our results indicate that single-molecule RT-PCR can be efficiently carried out in agarose matrix. Single-cell RT-PCR was successfully performed which showed a clear difference in gene expression level of EpCAM, a cancer biomarker gene, at the single-cell level between different types of cancer cells. This work clearly demonstrates for the first time, single-copy RT-PCR in agarose droplets. We believe this will open up new possibilities for viral RNA detection and single-cell transcription analysis.

Printing 2-dimentional droplet array for single-cell reverse transcription quantitative PCR assay with a microfluidic robot.

PubMed

Zhu, Ying; Zhang, Yun-Xia; Liu, Wen-Wen; Ma, Yan; Fang, Qun; Yao, Bo

2015-04-01

This paper describes a nanoliter droplet array-based single-cell reverse transcription quantitative PCR (RT-qPCR) assay method for quantifying gene expression in individual cells. By sequentially printing nanoliter-scale droplets on microchip using a microfluidic robot, all liquid-handling operations including cell encapsulation, lysis, reverse transcription, and quantitative PCR with real-time fluorescence detection, can be automatically achieved. The inhibition effect of cell suspension buffer on RT-PCR assay was comprehensively studied to achieve high-sensitivity gene quantification. The present system was applied in the quantitative measurement of expression level of mir-122 in single Huh-7 cells. A wide distribution of mir-122 expression in single cells from 3061 copies/cell to 79998 copies/cell was observed, showing a high level of cell heterogeneity. With the advantages of full-automation in liquid-handling, simple system structure, and flexibility in achieving multi-step operations, the present method provides a novel liquid-handling mode for single cell gene expression analysis, and has significant potentials in transcriptional identification and rare cell analysis.

Printing 2-Dimentional Droplet Array for Single-Cell Reverse Transcription Quantitative PCR Assay with a Microfluidic Robot

PubMed Central

Zhu, Ying; Zhang, Yun-Xia; Liu, Wen-Wen; Ma, Yan; Fang, Qun; Yao, Bo

2015-01-01

This paper describes a nanoliter droplet array-based single-cell reverse transcription quantitative PCR (RT-qPCR) assay method for quantifying gene expression in individual cells. By sequentially printing nanoliter-scale droplets on microchip using a microfluidic robot, all liquid-handling operations including cell encapsulation, lysis, reverse transcription, and quantitative PCR with real-time fluorescence detection, can be automatically achieved. The inhibition effect of cell suspension buffer on RT-PCR assay was comprehensively studied to achieve high-sensitivity gene quantification. The present system was applied in the quantitative measurement of expression level of mir-122 in single Huh-7 cells. A wide distribution of mir-122 expression in single cells from 3061 copies/cell to 79998 copies/cell was observed, showing a high level of cell heterogeneity. With the advantages of full-automation in liquid-handling, simple system structure, and flexibility in achieving multi-step operations, the present method provides a novel liquid-handling mode for single cell gene expression analysis, and has significant potentials in transcriptional identification and rare cell analysis. PMID:25828383

Single-cell optoporation and transfection using femtosecond laser and optical tweezers.

PubMed

Waleed, Muhammad; Hwang, Sun-Uk; Kim, Jung-Dae; Shabbir, Irfan; Shin, Sang-Mo; Lee, Yong-Gu

2013-01-01

In this paper, we demonstrate a new single-cell optoporation and transfection technique using a femtosecond Gaussian laser beam and optical tweezers. Tightly focused near-infrared (NIR) femtosecond laser pulse was employed to transiently perforate the cellular membrane at a single point in MCF-7 cancer cells. A distinct technique was developed by trapping the microparticle using optical tweezers to focus the femtosecond laser precisely on the cell membrane to puncture it. Subsequently, an external gene was introduced in the cell by trapping and inserting the same plasmid-coated microparticle into the optoporated cell using optical tweezers. Various experimental parameters such as femtosecond laser exposure power, exposure time, puncture hole size, exact focusing of the femtosecond laser on the cell membrane, and cell healing time were closely analyzed to create the optimal conditions for cell viability. Following the insertion of plasmid-coated microparticles in the cell, the targeted cells exhibited green fluorescent protein (GFP) under the fluorescent microscope, hence confirming successful transfection into the cell. This new optoporation and transfection technique maximizes the level of selectivity and control over the targeted cell, and this may be a breakthrough method through which to induce controllable genetic changes in the cell.

Palladium-based Mass-Tag Cell Barcoding with a Doublet-Filtering Scheme and Single Cell Deconvolution Algorithm

PubMed Central

Zunder, Eli R.; Finck, Rachel; Behbehani, Gregory K.; Amir, El-ad D.; Krishnaswamy, Smita; Gonzalez, Veronica D.; Lorang, Cynthia G.; Bjornson, Zach; Spitzer, Matthew H.; Bodenmiller, Bernd; Fantl, Wendy J.; Pe’er, Dana; Nolan, Garry P.

2015-01-01

SUMMARY Mass-tag cell barcoding (MCB) labels individual cell samples with unique combinatorial barcodes, after which they are pooled for processing and measurement as a single multiplexed sample. The MCB method eliminates variability between samples in antibody staining and instrument sensitivity, reduces antibody consumption, and shortens instrument measurement time. Here, we present an optimized MCB protocol with several improvements over previously described methods. The use of palladium-based labeling reagents expands the number of measurement channels available for mass cytometry and reduces interference with lanthanide-based antibody measurement. An error-detecting combinatorial barcoding scheme allows cell doublets to be identified and removed from the analysis. A debarcoding algorithm that is single cell-based rather than population-based improves the accuracy and efficiency of sample deconvolution. This debarcoding algorithm has been packaged into software that allows rapid and unbiased sample deconvolution. The MCB procedure takes 3–4 h, not including sample acquisition time of ~1 h per million cells. PMID:25612231

The ultimate picture-the combination of live cell superresolution microscopy and single molecule tracking yields highest spatio-temporal resolution.

PubMed

Dersch, Simon; Graumann, Peter L

2018-06-01

We are witnessing a breathtaking development in light (fluorescence) microscopy, where structures can be resolved down to the size of a ribosome within cells. This has already yielded surprising insight into the subcellular structure of cells, including the smallest cells, bacteria. Moreover, it has become possible to visualize and track single fluorescent protein fusions in real time, and quantify molecule numbers within individual cells. Combined, super resolution and single molecule tracking are pushing the limits of our understanding of the spatio-temporal organization even of the smallest cells to an unprecedented depth. Copyright © 2017 Elsevier Ltd. All rights reserved.

Single-cell analysis of S. cerevisiae growth recovery after a sublethal heat-stress applied during an alcoholic fermentation.

PubMed

Tibayrenc, Pierre; Preziosi-Belloy, Laurence; Ghommidh, Charles

2011-06-01

Interest in bioethanol production has experienced a resurgence in the last few years. Poor temperature control in industrial fermentation tanks exposes the yeast cells used for this production to intermittent heat stress which impairs fermentation efficiency. Therefore, there is a need for yeast strains with improved tolerance, able to recover from such temperature variations. Accordingly, this paper reports the development of methods for the characterization of Saccharomyces cerevisiae growth recovery after a sublethal heat stress. Single-cell measurements were carried out in order to detect cell-to-cell variability. Alcoholic batch fermentations were performed on a defined medium in a 2 l instrumented bioreactor. A rapid temperature shift from 33 to 43 °C was applied when ethanol concentration reached 50 g l⁻¹. Samples were collected at different times after the temperature shift. Single cell growth capability, lag-time and initial growth rate were determined by monitoring the growth of a statistically significant number of cells after agar medium plating. The rapid temperature shift resulted in an immediate arrest of growth and triggered a progressive loss of cultivability from 100 to 0.0001% within 8 h. Heat-injured cells were able to recover their growth capability on agar medium after a lag phase. Lag-time was longer and more widely distributed as the time of heat exposure increased. Thus, lag-time distribution gives an insight into strain sensitivity to heat-stress, and could be helpful for the selection of yeast strains of technological interest.

Live single cell functional phenotyping in droplet nano-liter reactors

NASA Astrophysics Data System (ADS)

Konry, Tania; Golberg, Alexander; Yarmush, Martin

2013-11-01

While single cell heterogeneity is present in all biological systems, most studies cannot address it due to technical limitations. Here we describe a nano-liter droplet microfluidic-based approach for stimulation and monitoring of surfaceand secreted markers of live single immune dendritic cells (DCs) as well as monitoring the live T cell/DC interaction. This nano-liter in vivo simulating microenvironment allows delivering various stimuli reagents to each cell and appropriate gas exchanges which are necessary to ensure functionality and viability of encapsulated cells. Labeling bioassay and microsphere sensors were integrated into nano-liter reaction volume of the droplet to monitor live single cell surface markers and secretion analysis in the time-dependent fashion. Thus live cell stimulation, secretion and surface monitoring can be obtained simultaneously in distinct microenvironment, which previously was possible using complicated and multi-step in vitro and in vivo live-cell microscopy, together with immunological studies of the outcome secretion of cellular function.

Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy

PubMed Central

Mazo-Vargas, Anyimilehidi; Park, Heungwon; Aydin, Mert; Buchler, Nicolas E.

2014-01-01

Time-lapse fluorescence microscopy is an important tool for measuring in vivo gene dynamics in single cells. However, fluorescent proteins are limited by slow chromophore maturation times and the cellular autofluorescence or phototoxicity that arises from light excitation. An alternative is luciferase, an enzyme that emits photons and is active upon folding. The photon flux per luciferase is significantly lower than that for fluorescent proteins. Thus time-lapse luminescence microscopy has been successfully used to track gene dynamics only in larger organisms and for slower processes, for which more total photons can be collected in one exposure. Here we tested green, yellow, and red beetle luciferases and optimized substrate conditions for in vivo luminescence. By combining time-lapse luminescence microscopy with a microfluidic device, we tracked the dynamics of cell cycle genes in single yeast with subminute exposure times over many generations. Our method was faster and in cells with much smaller volumes than previous work. Fluorescence of an optimized reporter (Venus) lagged luminescence by 15–20 min, which is consistent with its known rate of chromophore maturation in yeast. Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression. PMID:25232010

Improved Time-Lapsed Angular Scattering Microscopy of Single Cells

NASA Astrophysics Data System (ADS)

Cannaday, Ashley E.

By measuring angular scattering patterns from biological samples and fitting them with a Mie theory model, one can estimate the organelle size distribution within many cells. Quantitative organelle sizing of ensembles of cells using this method has been well established. Our goal is to develop the methodology to extend this approach to the single cell level, measuring the angular scattering at multiple time points and estimating the non-nuclear organelle size distribution parameters. The diameters of individual organelle-size beads were successfully extracted using scattering measurements with a minimum deflection angle of 20 degrees. However, the accuracy of size estimates can be limited by the angular range detected. In particular, simulations by our group suggest that, for cell organelle populations with a broader size distribution, the accuracy of size prediction improves substantially if the minimum angle of detection angle is 15 degrees or less. The system was therefore modified to collect scattering angles down to 10 degrees. To confirm experimentally that size predictions will become more stable when lower scattering angles are detected, initial validations were performed on individual polystyrene beads ranging in diameter from 1 to 5 microns. We found that the lower minimum angle enabled the width of this delta-function size distribution to be predicted more accurately. Scattering patterns were then acquired and analyzed from single mouse squamous cell carcinoma cells at multiple time points. The scattering patterns exhibit angular dependencies that look unlike those of any single sphere size, but are well-fit by a broad distribution of sizes, as expected. To determine the fluctuation level in the estimated size distribution due to measurement imperfections alone, formaldehyde-fixed cells were measured. Subsequent measurements on live (non-fixed) cells revealed an order of magnitude greater fluctuation in the estimated sizes compared to fixed cells. With our improved and better-understood approach to single cell angular scattering, we are now capable of reliably detecting changes in organelle size predictions due to biological causes above our measurement error of 20 nm, which enables us to apply our system to future studies of the investigation of various single cell biological processes.

Oxygen Nanobubble Tracking by Light Scattering in Single Cells and Tissues.

PubMed

Bhandari, Pushpak; Wang, Xiaolei; Irudayaraj, Joseph

2017-03-28

Oxygen nanobubbles (ONBs) have significant potential in targeted imaging and treatment in cancer diagnosis and therapy. Precise localization and tracking of single ONBs is demonstrated based on hyperspectral dark-field microscope (HSDFM) to image and track single oxygen nanobubbles in single cells. ONBs were proposed as promising contrast-generating imaging agents due to their strong light scattering generated from nonuniformity of refractive index at the interface. With this powerful platform, we have revealed the trajectories and quantities of ONBs in cells, and demonstrated the relation between the size and diffusion coefficient. We have also evaluated the presence of ONBs in the nucleus with respect to an increase in incubation time and have quantified the uptake in single cells in ex vivo tumor tissues. Our results demonstrate that HSDFM can be a versatile platform to detect and measure cellulosic nanoparticles at the single-cell level and to assess the dynamics and trajectories of this delivery system.

In vivo and in situ monitoring of the nitric oxide stimulus response of single cancer cells by Raman spectroscopy

NASA Astrophysics Data System (ADS)

Su, L.; Chen, Y.; Zhang, G. N.; Wang, L. H.; Shen, A. G.; Zhou, X. D.; Wang, X. H.; Hu, J. M.

2013-04-01

Raman spectroscopy is capable of studying time-resolved information of selected biomolecular distributions inside individual cells without labeling. In this study, Raman spectroscopy was for the first time utilized to in vivo and in situ monitor the cellular response to nitric oxide (NO) in single oral squamous cell carcinoma (OSCC) cells over a period of 24 h. Sodium nitroprusside (SNP) was chosen as a NO donor to be incubated with the OSCC cell line (TCA8113) for certain time intervals. In vivo and in situ Raman analysis revealed that the degradation and conformational changes of nucleic acids, lipids and proteins could be directly observed by changes in the characteristic Raman bands. In comparison with conventional flow cytometric analysis, Raman spectroscopy not only detected more subtle NO-induced chemical changes of cells, where the SNP concentration could be even less than 1 mM, but also provided a full view of the whole chemical components of single cells. Raman spectroscopy therefore is an important candidate for label-free, nondestructive and in situ monitoring of cellular changes in response to chemotherapeutic agents, which could potentially be used in rapid screening of novel drugs.

Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs.

PubMed

Hayashi, Tetsutaro; Ozaki, Haruka; Sasagawa, Yohei; Umeda, Mana; Danno, Hiroki; Nikaido, Itoshi

2018-02-12

Total RNA sequencing has been used to reveal poly(A) and non-poly(A) RNA expression, RNA processing and enhancer activity. To date, no method for full-length total RNA sequencing of single cells has been developed despite the potential of this technology for single-cell biology. Here we describe random displacement amplification sequencing (RamDA-seq), the first full-length total RNA-sequencing method for single cells. Compared with other methods, RamDA-seq shows high sensitivity to non-poly(A) RNA and near-complete full-length transcript coverage. Using RamDA-seq with differentiation time course samples of mouse embryonic stem cells, we reveal hundreds of dynamically regulated non-poly(A) transcripts, including histone transcripts and long noncoding RNA Neat1. Moreover, RamDA-seq profiles recursive splicing in >300-kb introns. RamDA-seq also detects enhancer RNAs and their cell type-specific activity in single cells. Taken together, we demonstrate that RamDA-seq could help investigate the dynamics of gene expression, RNA-processing events and transcriptional regulation in single cells.

Identifying stochastic oscillations in single-cell live imaging time series using Gaussian processes

PubMed Central

Manning, Cerys; Rattray, Magnus

2017-01-01

Multiple biological processes are driven by oscillatory gene expression at different time scales. Pulsatile dynamics are thought to be widespread, and single-cell live imaging of gene expression has lead to a surge of dynamic, possibly oscillatory, data for different gene networks. However, the regulation of gene expression at the level of an individual cell involves reactions between finite numbers of molecules, and this can result in inherent randomness in expression dynamics, which blurs the boundaries between aperiodic fluctuations and noisy oscillators. This underlies a new challenge to the experimentalist because neither intuition nor pre-existing methods work well for identifying oscillatory activity in noisy biological time series. Thus, there is an acute need for an objective statistical method for classifying whether an experimentally derived noisy time series is periodic. Here, we present a new data analysis method that combines mechanistic stochastic modelling with the powerful methods of non-parametric regression with Gaussian processes. Our method can distinguish oscillatory gene expression from random fluctuations of non-oscillatory expression in single-cell time series, despite peak-to-peak variability in period and amplitude of single-cell oscillations. We show that our method outperforms the Lomb-Scargle periodogram in successfully classifying cells as oscillatory or non-oscillatory in data simulated from a simple genetic oscillator model and in experimental data. Analysis of bioluminescent live-cell imaging shows a significantly greater number of oscillatory cells when luciferase is driven by a Hes1 promoter (10/19), which has previously been reported to oscillate, than the constitutive MoMuLV 5’ LTR (MMLV) promoter (0/25). The method can be applied to data from any gene network to both quantify the proportion of oscillating cells within a population and to measure the period and quality of oscillations. It is publicly available as a MATLAB package. PMID:28493880

Sequencing Single Cell Microbial Genomes with Microfluidic Amplifications Tools (MICW - Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

ScienceCinema

Quake, Steve

2018-02-02

Stanford University's Steve Quake on "Sequencing Single Cell Microbial Genomes with Microfluidic Amplification Tools" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

Sequencing Single Cell Microbial Genomes with Microfluidic Amplifications Tools (MICW - Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Quake, Steve

2011-10-12

Stanford University's Steve Quake on "Sequencing Single Cell Microbial Genomes with Microfluidic Amplification Tools" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

DRUG-NEM: Optimizing drug combinations using single-cell perturbation response to account for intratumoral heterogeneity

PubMed Central

Anchang, Benedict; Davis, Kara L.; Fienberg, Harris G.; Bendall, Sean C.; Karacosta, Loukia G.; Tibshirani, Robert; Nolan, Garry P.; Plevritis, Sylvia K.

2018-01-01

An individual malignant tumor is composed of a heterogeneous collection of single cells with distinct molecular and phenotypic features, a phenomenon termed intratumoral heterogeneity. Intratumoral heterogeneity poses challenges for cancer treatment, motivating the need for combination therapies. Single-cell technologies are now available to guide effective drug combinations by accounting for intratumoral heterogeneity through the analysis of the signaling perturbations of an individual tumor sample screened by a drug panel. In particular, Mass Cytometry Time-of-Flight (CyTOF) is a high-throughput single-cell technology that enables the simultaneous measurements of multiple (>40) intracellular and surface markers at the level of single cells for hundreds of thousands of cells in a sample. We developed a computational framework, entitled Drug Nested Effects Models (DRUG-NEM), to analyze CyTOF single-drug perturbation data for the purpose of individualizing drug combinations. DRUG-NEM optimizes drug combinations by choosing the minimum number of drugs that produce the maximal desired intracellular effects based on nested effects modeling. We demonstrate the performance of DRUG-NEM using single-cell drug perturbation data from tumor cell lines and primary leukemia samples. PMID:29654148

Single-cell measurement of red blood cell oxygen affinity.

PubMed

Di Caprio, Giuseppe; Stokes, Chris; Higgins, John M; Schonbrun, Ethan

2015-08-11

Oxygen is transported throughout the body by hemoglobin (Hb) in red blood cells (RBCs). Although the oxygen affinity of blood is well-understood and routinely assessed in patients by pulse oximetry, variability at the single-cell level has not been previously measured. In contrast, single-cell measurements of RBC volume and Hb concentration are taken millions of times per day by clinical hematology analyzers, and they are important factors in determining the health of the hematologic system. To better understand the variability and determinants of oxygen affinity on a cellular level, we have developed a system that quantifies the oxygen saturation, cell volume, and Hb concentration for individual RBCs in high throughput. We find that the variability in single-cell saturation peaks at an oxygen partial pressure of 2.9%, which corresponds to the maximum slope of the oxygen-Hb dissociation curve. In addition, single-cell oxygen affinity is positively correlated with Hb concentration but independent of osmolarity, which suggests variation in the Hb to 2,3-diphosphoglycerate (2-3 DPG) ratio on a cellular level. By quantifying the functional behavior of a cellular population, our system adds a dimension to blood cell analysis and other measurements of single-cell variability.

Single-cell measurement of red blood cell oxygen affinity

PubMed Central

Di Caprio, Giuseppe; Stokes, Chris; Higgins, John M.; Schonbrun, Ethan

2015-01-01

Oxygen is transported throughout the body by hemoglobin (Hb) in red blood cells (RBCs). Although the oxygen affinity of blood is well-understood and routinely assessed in patients by pulse oximetry, variability at the single-cell level has not been previously measured. In contrast, single-cell measurements of RBC volume and Hb concentration are taken millions of times per day by clinical hematology analyzers, and they are important factors in determining the health of the hematologic system. To better understand the variability and determinants of oxygen affinity on a cellular level, we have developed a system that quantifies the oxygen saturation, cell volume, and Hb concentration for individual RBCs in high throughput. We find that the variability in single-cell saturation peaks at an oxygen partial pressure of 2.9%, which corresponds to the maximum slope of the oxygen–Hb dissociation curve. In addition, single-cell oxygen affinity is positively correlated with Hb concentration but independent of osmolarity, which suggests variation in the Hb to 2,3-diphosphoglycerate (2–3 DPG) ratio on a cellular level. By quantifying the functional behavior of a cellular population, our system adds a dimension to blood cell analysis and other measurements of single-cell variability. PMID:26216973

Protein adsorption in microengraving immunoassays.

PubMed

Song, Qing

2015-10-16

Microengraving is a novel immunoassay for characterizing multiple protein secretions from single cells. During the immunoassay, characteristic diffusion and kinetic time scales  and  determine the time for molecular diffusion of proteins secreted from the activated single lymphocytes and subsequent binding onto the glass slide surface respectively. Our results demonstrate that molecular diffusion plays important roles in the early stage of protein adsorption dynamics which shifts to a kinetic controlled mechanism in the later stage. Similar dynamic pathways are observed for protein adsorption with significantly fast rates and rapid shifts in transport mechanisms when  is increased a hundred times from 0.313 to 31.3. Theoretical adsorption isotherms follow the trend of experimentally obtained data. Adsorption isotherms indicate that amount of proteins secreted from individual cells and subsequently captured on a clean glass slide surface increases monotonically with time. Our study directly validates that protein secretion rates can be quantified by the microengraving immunoassay. This will enable us to apply microengraving immunoassays to quantify secretion rates from 10⁴-10⁵ single cells in parallel, screen antigen-specific cells with the highest secretion rate for clonal expansion and quantitatively reveal cellular heterogeneity within a small cell sample.

Protein Adsorption in Microengraving Immunoassays

PubMed Central

Song, Qing

2015-01-01

Microengraving is a novel immunoassay forcharacterizing multiple protein secretions from single cells. During the immunoassay, characteristic diffusion and kinetic time scales τD and τK determine the time for molecular diffusion of proteins secreted from the activated single lymphocytes and subsequent binding onto the glass slide surface respectively. Our results demonstrate that molecular diffusion plays important roles in the early stage of protein adsorption dynamics which shifts to a kinetic controlled mechanism in the later stage. Similar dynamic pathways are observed for protein adsorption with significantly fast rates and rapid shifts in transport mechanisms when C0* is increased a hundred times from 0.313 to 31.3. Theoretical adsorption isotherms follow the trend of experimentally obtained data. Adsorption isotherms indicate that amount of proteins secreted from individual cells and subsequently captured on a clean glass slide surface increases monotonically with time. Our study directly validates that protein secretion rates can be quantified by the microengraving immunoassay. This will enable us to apply microengraving immunoassays to quantify secretion rates from 104–105 single cells in parallel, screen antigen-specific cells with the highest secretion rate for clonal expansion and quantitatively reveal cellular heterogeneity within a small cell sample. PMID:26501282

Using single cell sequencing data to model the evolutionary history of a tumor.

PubMed

Kim, Kyung In; Simon, Richard

2014-01-24

The introduction of next-generation sequencing (NGS) technology has made it possible to detect genomic alterations within tumor cells on a large scale. However, most applications of NGS show the genetic content of mixtures of cells. Recently developed single cell sequencing technology can identify variation within a single cell. Characterization of multiple samples from a tumor using single cell sequencing can potentially provide information on the evolutionary history of that tumor. This may facilitate understanding how key mutations accumulate and evolve in lineages to form a heterogeneous tumor. We provide a computational method to infer an evolutionary mutation tree based on single cell sequencing data. Our approach differs from traditional phylogenetic tree approaches in that our mutation tree directly describes temporal order relationships among mutation sites. Our method also accommodates sequencing errors. Furthermore, we provide a method for estimating the proportion of time from the earliest mutation event of the sample to the most recent common ancestor of the sample of cells. Finally, we discuss current limitations on modeling with single cell sequencing data and possible improvements under those limitations. Inferring the temporal ordering of mutational sites using current single cell sequencing data is a challenge. Our proposed method may help elucidate relationships among key mutations and their role in tumor progression.

TLM-Tracker: software for cell segmentation, tracking and lineage analysis in time-lapse microscopy movies.

PubMed

Klein, Johannes; Leupold, Stefan; Biegler, Ilona; Biedendieck, Rebekka; Münch, Richard; Jahn, Dieter

2012-09-01

Time-lapse imaging in combination with fluorescence microscopy techniques enable the investigation of gene regulatory circuits and uncovered phenomena like culture heterogeneity. In this context, computational image processing for the analysis of single cell behaviour plays an increasing role in systems biology and mathematical modelling approaches. Consequently, we developed a software package with graphical user interface for the analysis of single bacterial cell behaviour. A new software called TLM-Tracker allows for the flexible and user-friendly interpretation for the segmentation, tracking and lineage analysis of microbial cells in time-lapse movies. The software package, including manual, tutorial video and examples, is available as Matlab code or executable binaries at http://www.tlmtracker.tu-bs.de.

Label-free cell-cycle analysis by high-throughput quantitative phase time-stretch imaging flow cytometry

NASA Astrophysics Data System (ADS)

Mok, Aaron T. Y.; Lee, Kelvin C. M.; Wong, Kenneth K. Y.; Tsia, Kevin K.

2018-02-01

Biophysical properties of cells could complement and correlate biochemical markers to characterize a multitude of cellular states. Changes in cell size, dry mass and subcellular morphology, for instance, are relevant to cell-cycle progression which is prevalently evaluated by DNA-targeted fluorescence measurements. Quantitative-phase microscopy (QPM) is among the effective biophysical phenotyping tools that can quantify cell sizes and sub-cellular dry mass density distribution of single cells at high spatial resolution. However, limited camera frame rate and thus imaging throughput makes QPM incompatible with high-throughput flow cytometry - a gold standard in multiparametric cell-based assay. Here we present a high-throughput approach for label-free analysis of cell cycle based on quantitative-phase time-stretch imaging flow cytometry at a throughput of > 10,000 cells/s. Our time-stretch QPM system enables sub-cellular resolution even at high speed, allowing us to extract a multitude (at least 24) of single-cell biophysical phenotypes (from both amplitude and phase images). Those phenotypes can be combined to track cell-cycle progression based on a t-distributed stochastic neighbor embedding (t-SNE) algorithm. Using multivariate analysis of variance (MANOVA) discriminant analysis, cell-cycle phases can also be predicted label-free with high accuracy at >90% in G1 and G2 phase, and >80% in S phase. We anticipate that high throughput label-free cell cycle characterization could open new approaches for large-scale single-cell analysis, bringing new mechanistic insights into complex biological processes including diseases pathogenesis.

Fast Raman single bacteria identification: toward a routine in-vitro diagnostic

NASA Astrophysics Data System (ADS)

Douet, Alice; Josso, Quentin; Marchant, Adrien; Dutertre, Bertrand; Filiputti, Delphine; Novelli-Rousseau, Armelle; Espagnon, Isabelle; Kloster-Landsberg, Meike; Mallard, Frédéric; Perraut, Francois

2016-04-01

Timely microbiological results are essential to allow clinicians to optimize the prescribed treatment, ideally at the initial stage of the therapeutic process. Several approaches have been proposed to solve this issue and to provide the microbiological result in a few hours directly from the sample such as molecular biology. However fast and sensitive those methods are not based on single phenotypic information which presents several drawbacks and limitations. Optical methods have the advantage to allow single-cell sensitivity and to probe the phenotype of measured cells. Here we present a process and a prototype that allow automated single-bacteria phenotypic analysis. This prototype is based on the use of Digital In-line Holography techniques combined with a specially designed Raman spectrometer using a dedicated device to capture bacteria. The localization of single-cell is finely determined by using holograms and a proper propagation kernel. Holographic images are also used to analyze bacteria in the sample to sort potential pathogens from flora dwelling species or other biological particles. This accurate localization enables the use of a small confocal volume adapted to the measurement of single-cell. Along with the confocal volume adaptation, we also have modified every components of the spectrometer to optimize single-bacteria Raman measurements. This optimization allowed us to acquire informative single-cell spectra using an integration time of 0.5s only. Identification results obtained with this prototype are presented based on a 65144 Raman spectra database acquired automatically on 48 bacteria strains belonging to 8 species.

Time-lapse evaluation of human embryo development in single versus sequential culture media--a sibling oocyte study.

PubMed

Ciray, Haydar Nadir; Aksoy, Turan; Goktas, Cihan; Ozturk, Bilgen; Bahceci, Mustafa

2012-09-01

To compare the dynamics of early development between embryos cultured in single and sequential media. Randomized, comparative study. Private IVF centre. A total of 446 metaphase II oocytes from 51 couples who underwent oocyte retrieval procedure for intracytoplasmic sperm injection. Forty-nine resulted in embryo transfer. Oocytes were split between single and sequential media produced by the same manufacturer and cultured in a time-lapse incubator. Morphokinetic parameters until the embryos reached the 5-cell stage (t5), utilization, clinical pregnancy and implantation rates. Embryos cultured in single media were advanced from the first mitosis cycle and reached 2- to 5-cell stages earlier. There was not any difference between the durations for cell cycle two (cc2 = t3-t2) and s2 (t4-t3). The utilization, clinical pregnancy and implantation rates did not differ between groups. The proportion of cryopreserved day 6 embryos to two pronuclei oocytes was significantly higher in sequential than in single media. Morphokinetics of embryo development vary between single and sequential culture media at least until the 5-cell stage. The overall clinical and embryological parameters remain similar regardless of the culture system.

A novel method for multiparameter physiological phenotype characterization at the single-cell level

NASA Astrophysics Data System (ADS)

Kelbauskas, Laimonas; Ashili, Shashanka; Houkal, Jeff; Smith, Dean; Mohammadreza, Aida; Lee, Kristen; Kumar, Ashok; Anis, Yasser; Paulson, Tom; Youngbull, Cody; Tian, Yanqing; Johnson, Roger; Holl, Mark; Meldrum, Deirdre

2011-02-01

Non-genetic intercellular heterogeneity has been increasingly recognized as one of the key factors in a variety of core cellular processes including proliferation, stimulus response, carcinogenesis and drug resistance. Many diseases, including cancer, originate in a single or a few cells. Early detection and characterization of these abnormal cells can provide new insights into the pathogenesis and serve as a tool for better disease diagnosis and treatment. We report on a novel technology for multiparameter physiological phenotype characterization at the single-cell level. It is based on real-time measurements of concentrations of several metabolites by means of extracellular optical sensors in microchambers of sub-nL volume containing single cells. In its current configuration, the measurement platform features the capability to detect oxygen consumption rate and pH changes under normoxic and hypoxic conditions at the single-cell level. We have conceived, designed and developed a semi-automated method for single-cell manipulation and loading into microwells utilizing custom, high-precision fluid handling at the nanoliter scale. We present the results of a series of measurements of oxygen consumption rates (OCRs) of single human metaplastic esophageal epithelial cells. In addition, to assess the effects of cell-to-cell interactions, we have measured OCRs of two and three cells placed in a single well. The major advantages of the approach are a) multiplexed characterization of cell phenotype at the single-cell level, b) minimal invasiveness due to the distant positioning of sensors, and c) flexibility in terms of accommodating measurements of other metabolites or biomolecules of interest.

Nanotube antibody biosensor arrays for the detection of circulating breast cancer cells

NASA Astrophysics Data System (ADS)

Shao, Ning; Wickstrom, Eric; Panchapakesan, Balaji

2008-11-01

Recent reports have shown that nanoscale electronic devices can be used to detect a change in electrical properties when receptor proteins bind to their corresponding antibodies functionalized on the surface of the device, in extracts from as few as ten lysed tumor cells. We hypothesized that nanotube-antibody devices could sensitively and specifically detect entire live cancer cells. We report for the first time a single nanotube field effect transistor array, functionalized with IGF1R-specific and Her2-specific antibodies, which exhibits highly sensitive and selective sensing of live, intact MCF7 and BT474 human breast cancer cells in human blood. Those two cell lines both overexpress IGF1R and Her2, at different levels. Single or small bundle of nanotube devices that were functionalized with IGF1R-specific or Her2-specific antibodies showed 60% decreases in conductivity upon interaction with BT474 or MCF7 breast cancer cells in two µl drops of blood. Control experiments with non-specific antibodies or with MCF10A control breast cells produced a less than 5% decrease in electrical conductivity, illustrating the high sensitivity for whole cell binding by these single nanotube-antibody devices. We postulate that the free energy change due to multiple simultaneous cell-antibody binding events exerted stress along the nanotube surface, decreasing its electrical conductivity due to an increase in band gap. Because the free energy change upon cell-antibody binding, the stress exerted on the nanotube, and the change in conductivity are specific to a specific antigen-antibody interaction; these properties might be used as a fingerprint for the molecular sensing of circulating cancer cells. From optical microscopy observations during sensing, it appears that the binding of a single cell to a single nanotube field effect transistor produced the change in electrical conductivity. Thus we report a nanoscale oncometer with single cell sensitivity with a diameter 1000 times smaller than a cancer cell that functions in a drop of fresh blood.

Stationary nanoliter droplet array with a substrate of choice for single adherent/nonadherent cell incubation and analysis.

PubMed

Shemesh, Jonathan; Ben Arye, Tom; Avesar, Jonathan; Kang, Joo H; Fine, Amir; Super, Michael; Meller, Amit; Ingber, Donald E; Levenberg, Shulamit

2014-08-05

Microfluidic water-in-oil droplets that serve as separate, chemically isolated compartments can be applied for single-cell analysis; however, to investigate encapsulated cells effectively over prolonged time periods, an array of droplets must remain stationary on a versatile substrate for optimal cell compatibility. We present here a platform of unique geometry and substrate versatility that generates a stationary nanodroplet array by using wells branching off a main microfluidic channel. These droplets are confined by multiple sides of a nanowell and are in direct contact with a biocompatible substrate of choice. The device is operated by a unique and reversed loading procedure that eliminates the need for fine pressure control or external tubing. Fluorocarbon oil isolates the droplets and provides soluble oxygen for the cells. By using this approach, the metabolic activity of single adherent cells was monitored continuously over time, and the concentration of viable pathogens in blood-derived samples was determined directly by measuring the number of colony-formed droplets. The method is simple to operate, requires a few microliters of reagent volume, is portable, is reusable, and allows for cell retrieval. This technology may be particularly useful for multiplexed assays for which prolonged and simultaneous visual inspection of many isolated single adherent or nonadherent cells is required.

Stationary nanoliter droplet array with a substrate of choice for single adherent/nonadherent cell incubation and analysis

PubMed Central

Shemesh, Jonathan; Ben Arye, Tom; Avesar, Jonathan; Kang, Joo H.; Fine, Amir; Super, Michael; Meller, Amit; Ingber, Donald E.; Levenberg, Shulamit

2014-01-01

Microfluidic water-in-oil droplets that serve as separate, chemically isolated compartments can be applied for single-cell analysis; however, to investigate encapsulated cells effectively over prolonged time periods, an array of droplets must remain stationary on a versatile substrate for optimal cell compatibility. We present here a platform of unique geometry and substrate versatility that generates a stationary nanodroplet array by using wells branching off a main microfluidic channel. These droplets are confined by multiple sides of a nanowell and are in direct contact with a biocompatible substrate of choice. The device is operated by a unique and reversed loading procedure that eliminates the need for fine pressure control or external tubing. Fluorocarbon oil isolates the droplets and provides soluble oxygen for the cells. By using this approach, the metabolic activity of single adherent cells was monitored continuously over time, and the concentration of viable pathogens in blood-derived samples was determined directly by measuring the number of colony-formed droplets. The method is simple to operate, requires a few microliters of reagent volume, is portable, is reusable, and allows for cell retrieval. This technology may be particularly useful for multiplexed assays for which prolonged and simultaneous visual inspection of many isolated single adherent or nonadherent cells is required. PMID:25053808

Practical, microfabrication-free device for single-cell isolation.

PubMed

Lin, Liang-I; Chao, Shih-Hui; Meldrum, Deirdre R

2009-08-21

Microfabricated devices have great potential in cell-level studies, but are not easily accessible for the broad biology community. This paper introduces the Microscale Oil-Covered Cell Array (MOCCA) as a low-cost device for high throughput single-cell analysis that can be easily produced by researchers without microengineering knowledge. Instead of using microfabricated structures to capture cells, MOCCA isolates cells in discrete aqueous droplets that are separated by oil on patterned hydrophilic areas across a relatively more hydrophobic substrate. The number of randomly seeded Escherichia coli bacteria in each discrete droplet approaches single-cell levels. The cell distribution on MOCCA is well-fit with Poisson distribution. In this pioneer study, we created an array of 900-picoliter droplets. The total time needed to seed cells in approximately 3000 droplets was less than 10 minutes. Compared to traditional microfabrication techniques, MOCCA dramatically lowers the cost of microscale cell arrays, yet enhances the fabrication and operational efficiency for single-cell analysis.

Single Cell Proteolytic Assays to Investigate Cancer Clonal Heterogeneity and Cell Dynamics Using an Efficient Cell Loading Scheme

NASA Astrophysics Data System (ADS)

Chen, Yu-Chih; Cheng, Yu-Heng; Ingram, Patrick; Yoon, Euisik

2016-06-01

Proteolytic degradation of the extracellular matrix (ECM) is critical in cancer invasion, and recent work suggests that heterogeneous cancer populations cooperate in this process. Despite the importance of cell heterogeneity, conventional proteolytic assays measure average activity, requiring thousands of cells and providing limited information about heterogeneity and dynamics. Here, we developed a microfluidic platform that provides high-efficiency cell loading and simple valveless isolation, so the proteolytic activity of a small sample (10-100 cells) can be easily characterized. Combined with a single cell derived (clonal) sphere formation platform, we have successfully demonstrated the importance of microenvironmental cues for proteolytic activity and also investigated the difference between clones. Furthermore, the platform allows monitoring single cells at multiple time points, unveiling different cancer cell line dynamics in proteolytic activity. The presented tool facilitates single cell proteolytic analysis using small samples, and our findings illuminate the heterogeneous and dynamic nature of proteolytic activity.

Rapid acquisition of mean Raman spectra of eukaryotic cells for a robust single cell classification.

PubMed

Schie, Iwan W; Kiselev, Roman; Krafft, Christoph; Popp, Jürgen

2016-11-14

Raman spectroscopy has previously been used to identify eukaryotic and prokaryotic cells. While prokaryotic cells are small in size and can be assessed by a single Raman spectrum, the larger size of eukaryotic cells and their complex organization requires the acquisition of multiple Raman spectra to properly characterize them. A Raman spectrum from a diffraction-limited spot at an arbitrary location within a cell results in spectral variations that affect classification approaches. To probe whole cells with Raman imaging at high spatial resolution is time consuming, because a large number of Raman spectra need to be collected, resulting in low cell throughput and impairing statistical analysis due to low cell numbers. Here we propose a method to overcome the effects of cellular heterogeneity by acquiring integrated Raman spectra covering a large portion of a cell. The acquired spectrum represents the mean macromolecular composition of a cell with an exposure time that is comparable to acquisition of a single Raman spectrum. Data sets were collected from T lymphocyte Jurkat cells, and pancreatic cell lines Capan1 and MiaPaca2. Cell classification by support vector machines was compared for single spectra, spectra of images and integrated Raman spectra of cells. The integrated approach provides better and more stable prediction for individual cells, and in the current implementation, the mean macromolecular information of a cell can be acquired faster than with the acquisition of individual spectra from a comparable region. It is expected that this approach will have a major impact on the implementation of Raman based cell classification.

Reinforcement of integrin-mediated T-Lymphocyte adhesion by TNF-induced Inside-out Signaling

NASA Astrophysics Data System (ADS)

Li, Qian; Huth, Steven; Adam, Dieter; Selhuber-Unkel, Christine

2016-07-01

Integrin-mediated leukocyte adhesion to endothelial cells is a crucial step in immunity against pathogens. Whereas the outside-in signaling pathway in response to the pro-inflammatory cytokine tumour necrosis factor (TNF) has already been studied in detail, little knowledge exists about a supposed TNF-mediated inside-out signaling pathway. In contrast to the outside-in signaling pathway, which relies on the TNF-induced upregulation of surface molecules on endothelium, inside-out signaling should also be present in an endothelium-free environment. Using single-cell force spectroscopy, we show here that stimulating Jurkat cells with TNF significantly reinforces their adhesion to fibronectin in a biomimetic in vitro assay for cell-surface contact times of about 1.5 seconds, whereas for larger contact times the effect disappears. Analysis of single-molecule ruptures further demonstrates that TNF strengthens sub-cellular single rupture events at short cell-surface contact times. Hence, our results provide quantitative evidence for the significant impact of TNF-induced inside-out signaling in the T-lymphocyte initial adhesion machinery.

Report of the Microbial Development Working Group

NASA Technical Reports Server (NTRS)

Nelson, G.

1985-01-01

In formulating ideas on the relationship of gravity to the development, growth, and reproduction of microorganisms, a rather liberal definition of microorganisms is used which includes bacteria, yeasts, protists, filamentous fungi, and single cells in culture. A principal advantage of microorganisms as experimental subjects is the rigor with which they can be defined and controlled. As single cells, each cell may be regarded as identical to the others in the population. This property applies to the morphology, physiology, and genetic parameters of the cells. The growth and development of the population is subject to precise manipulation as the nutritional requirements are known and minimal media formulations have been developed. Growth and differentiation can be manipulated in a variety of ways, such as alteration of the culture temperature and food supply, or by use of mutants. Finally, the short generation times of microorganisms provide the opportunity to conduct multigenerational studies within practical time limits and, in a similar vein, cellular responses to various stimuli or stresses are conveniently monitored because of the rapid response times of single cells.

A statistical framework for multiparameter analysis at the single-cell level.

PubMed

Torres-García, Wandaliz; Ashili, Shashanka; Kelbauskas, Laimonas; Johnson, Roger H; Zhang, Weiwen; Runger, George C; Meldrum, Deirdre R

2012-03-01

Phenotypic characterization of individual cells provides crucial insights into intercellular heterogeneity and enables access to information that is unavailable from ensemble averaged, bulk cell analyses. Single-cell studies have attracted significant interest in recent years and spurred the development of a variety of commercially available and research-grade technologies. To quantify cell-to-cell variability of cell populations, we have developed an experimental platform for real-time measurements of oxygen consumption (OC) kinetics at the single-cell level. Unique challenges inherent to these single-cell measurements arise, and no existing data analysis methodology is available to address them. Here we present a data processing and analysis method that addresses challenges encountered with this unique type of data in order to extract biologically relevant information. We applied the method to analyze OC profiles obtained with single cells of two different cell lines derived from metaplastic and dysplastic human Barrett's esophageal epithelium. In terms of method development, three main challenges were considered for this heterogeneous dynamic system: (i) high levels of noise, (ii) the lack of a priori knowledge of single-cell dynamics, and (iii) the role of intercellular variability within and across cell types. Several strategies and solutions to address each of these three challenges are presented. The features such as slopes, intercepts, breakpoint or change-point were extracted for every OC profile and compared across individual cells and cell types. The results demonstrated that the extracted features facilitated exposition of subtle differences between individual cells and their responses to cell-cell interactions. With minor modifications, this method can be used to process and analyze data from other acquisition and experimental modalities at the single-cell level, providing a valuable statistical framework for single-cell analysis.

Micromechanical and surface adhesive properties of single saccharomyces cerevisiae cells

NASA Astrophysics Data System (ADS)

Farzi, Bahman; Cetinkaya, Cetin

2017-09-01

The adhesion and mechanical properties of a biological cell (e.g. cell membrane elasticity and adhesiveness) are often strong indicators for the state of its health. Many existing techniques for determining mechanical properties of cells require direct physical contact with a single cell or a group of cells. Physical contact with the cell can trigger complex mechanotransduction mechanisms, leading to cellular responses, and consequently interfering with measurement accuracy. In the current work, based on ultrasonic excitation and interferometric (optical) motion detection, a non-contact method for characterizing the adhesion and mechanical properties of single cells is presented. It is experimentally demonstrated that the rocking (rigid body) motion and internal vibrational resonance frequencies of a single saccharomyces cerevisiae (SC) (baker’s yeast) cell can be acquired with the current approach, and the Young’s modulus and surface tension of the cell membrane as well as surface adhesion energy can be extracted from the values of these acquired resonance frequencies. The detected resonance frequency ranges for single SC cells include a rocking (rigid body) frequency of 330  ±  70 kHz and two breathing resonance frequencies of 1.53  ±  0.12 and 2.02  ±  0.31 MHz. Based on these values, the average work-of-adhesion of SC cells on a silicon substrate in aqueous medium is extracted, for the first time, as WASC-Si=16.2+/- 3.8 mJ {{m}-2} . Similarly, the surface tension and the Young’s modulus of the SC cell wall are predicted as {{σ }SC}=0.16+/- 0.02 N {{m}-1} and {{E}SC}= 9.20  ±  2.80 MPa, respectively. These results are compared to those reported in the literature by utilizing various methods, and good agreements are found. The current approach eliminates the measurement inaccuracies associated with the physical contact. Exciting and detecting cell dynamics at micro-second time-scales is significantly faster than the currently known metabolistic response times of cells (milliseconds to seconds), thus, it has the potential to decouple metabolistic and mechanotransduction effects from external stimuli and to operate at high throughput rates.

Single molecule analysis of B cell receptor motion during signaling activation

NASA Astrophysics Data System (ADS)

Rey Suarez, Ivan; Koo, Peter; Zhou, Shu; Wheatley, Brittany; Song, Wenxia; Mochrie, Simon; Upadhyaya, Arpita

B cells are an essential part of the adaptive immune system. They patrol the body for signs of infection in the form of antigen on the surface of antigen presenting cells. B cell receptor (BCR) binding to antigen induces a signaling cascade that leads to B cell activation and spreading. During activation, BCR form signaling microclusters that later coalesce as the cell contracts. We have studied the dynamics of BCRs on activated murine primary B cells using single particle tracking. The tracks are analyzed using perturbation expectation-maximization (pEM), a systems-level analysis, which allows identification of different short-time diffusive states from single molecule tracks. We identified four dominant diffusive states, two of which correspond to BCRs interacting with signaling molecules. For wild-type cells, the number of BCR in signaling states increases as the cell spreads and then decreases during cell contraction. In contrast, cells lacking the actin regulatory protein, N-WASP, are unable to contract and BCRs remain in the signaling states for longer times. These observations indicate that actin cytoskeleton dynamics modulate BCR diffusion and clustering. Our results provide novel information regarding the timescale of interaction between BCR and signaling molecules.

Probing cellular heterogeneity in cytokine-secreting immune cells using droplet-based microfluidics.

PubMed

Chokkalingam, Venkatachalam; Tel, Jurjen; Wimmers, Florian; Liu, Xin; Semenov, Sergey; Thiele, Julian; Figdor, Carl G; Huck, Wilhelm T S

2013-12-21

Here, we present a platform to detect cytokine (IL-2, IFN-γ, TNF-α) secretion of single, activated T-cells in droplets over time. We use a novel droplet-based microfluidic approach to encapsulate cells in monodisperse agarose droplets together with functionalized cytokine-capture beads for subsequent binding and detection of secreted cytokines from single cells. This method allows high-throughput detection of cellular heterogeneity and maps subsets within cell populations with specific functions.

Comparison of Normalized and Unnormalized Single Cell and Population Assemblies (MICW - Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

ScienceCinema

Hugenholtz, Phil

2018-02-12

University of Queensland's Phil Hugenholtz on "Comparison of Normalized and Unnormalized Single Cell and Population Assemblies" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

Soft-state biomicrofluidic pulse generator for single cell analysis

NASA Astrophysics Data System (ADS)

Sabounchi, Poorya; Ionescu-Zanetti, Cristian; Chen, Roger; Karandikar, Manjiree; Seo, Jeonggi; Lee, Luke P.

2006-05-01

We present the design, fabrication, and characterization of a soft-state biomicrofluidic pulse generator for single cell analysis. Hydrodynamic cell trapping via lateral microfluidic junctions allows the trapping of single cells from a bulk suspension. Microfluidic injection sites adjacent to the cell-trapping channels enable the pulsed delivery of nanoliter volumes of biochemical reagent. We demonstrated the application and removal of reagent at a frequency of 10Hz with a rise time of less than 33ms and a reagent consumption rate of 0.2nL/s. It is shown that this system operates as a low-pass filter with a cutoff frequency of 7Hz.

High-throughput label-free screening of euglena gracilis with optofluidic time-stretch quantitative phase microscopy

NASA Astrophysics Data System (ADS)

Guo, Baoshan; Lei, Cheng; Ito, Takuro; Yaxiaer, Yalikun; Kobayashi, Hirofumi; Jiang, Yiyue; Tanaka, Yo; Ozeki, Yasuyuki; Goda, Keisuke

2017-02-01

The development of reliable, sustainable, and economical sources of alternative fuels is an important, but challenging goal for the world. As an alternative to liquid fossil fuels, microalgal biofuel is expected to play a key role in reducing the detrimental effects of global warming since microalgae absorb atmospheric CO2 via photosynthesis. Unfortunately, conventional analytical methods only provide population-averaged lipid contents and fail to characterize a diverse population of microalgal cells with single-cell resolution in a noninvasive and interference-free manner. Here we demonstrate high-throughput label-free single-cell screening of lipid-producing microalgal cells with optofluidic time-stretch quantitative phase microscopy. In particular, we use Euglena gracilis - an attractive microalgal species that produces wax esters (suitable for biodiesel and aviation fuel after refinement) within lipid droplets. Our optofluidic time-stretch quantitative phase microscope is based on an integration of a hydrodynamic-focusing microfluidic chip, an optical time-stretch phase-contrast microscope, and a digital image processor equipped with machine learning. As a result, it provides both the opacity and phase contents of every single cell at a high throughput of 10,000 cells/s. We characterize heterogeneous populations of E. gracilis cells under two different culture conditions to evaluate their lipid production efficiency. Our method holds promise as an effective analytical tool for microalgaebased biofuel production.

Cell-substrate impedance fluctuations of single amoeboid cells encode cell-shape and adhesion dynamics

NASA Astrophysics Data System (ADS)

Leonhardt, Helmar; Gerhardt, Matthias; Höppner, Nadine; Krüger, Kirsten; Tarantola, Marco; Beta, Carsten

2016-01-01

We show systematic electrical impedance measurements of single motile cells on microelectrodes. Wild-type cells and mutant strains were studied that differ in their cell-substrate adhesion strength. We recorded the projected cell area by time-lapse microscopy and observed irregular oscillations of the cell shape. These oscillations were correlated with long-term variations in the impedance signal. Superposed to these long-term trends, we observed fluctuations in the impedance signal. Their magnitude clearly correlated with the adhesion strength, suggesting that strongly adherent cells display more dynamic cell-substrate interactions.

Plasmonic-based nanoprobes for dynamic sensing of single tumor cells (Conference Presentation)

NASA Astrophysics Data System (ADS)

Chen, Zixuan

2017-02-01

We described here two plasmonic-based nanoprobes with purpose of imaging dynamic biologic process of single tumor cells. At first, we proposed a multi-modified core-shell gold@silver nanorods for real-time monitoring the entire autophagy process at single-cell level. Autophagy is vital for understanding the mechanisms of human pathologies, developing novel drugs and exploring approaches for autophagy controlling. The plasmon resonance scattering spectra of the nanoprobes was superoxide radicals (O2•-)-dependent, a major indicator of cell autophagy, and suitable for real-time monitoring at single-cell level. More importantly, with the introduction of `relay probe' operation, two types of O2•-regulating autophagy processes were successfully traced from the beginning to the end, and the possible mechanism was also proposed. According to our results, intracellular O2•- level controlled the autophagy process by mediating the autolysosome generation. Different starvation approaches can induce different autophagy processes, such as diverse steady state time-consuming. In addition, a plasmonic-based nanothermometer was prepared via dense thermosensitive polymer (pNIPAAm) capping on gold nanorods, of which the plasmon resonance spectra was linearly dependent on adjacent temperature. In this work, the white light transmitted dark-field illuminator was replaced by a laser total internal reflection dark-field microscope (LTIR-DFM) system in order to overcome the low-throughput and inexorable biological scattering background of DFM, as well as interference from mechanic noise, nanoprobe direction, optical system drift, etc. With this nanothermometer, we have successfully captured temporal biological thermal process (thermogenesis) occurred in single tumor cells, providing a new potential strategy for in-situ cellular analysis.

An on-chip imaging droplet-sorting system: a real-time shape recognition method to screen target cells in droplets with single cell resolution

NASA Astrophysics Data System (ADS)

Girault, Mathias; Kim, Hyonchol; Arakawa, Hisayuki; Matsuura, Kenji; Odaka, Masao; Hattori, Akihiro; Terazono, Hideyuki; Yasuda, Kenji

2017-01-01

A microfluidic on-chip imaging cell sorter has several advantages over conventional cell sorting methods, especially to identify cells with complex morphologies such as clusters. One of the remaining problems is how to efficiently discriminate targets at the species level without labelling. Hence, we developed a label-free microfluidic droplet-sorting system based on image recognition of cells in droplets. To test the applicability of this method, a mixture of two plankton species with different morphologies (Dunaliella tertiolecta and Phaeodactylum tricornutum) were successfully identified and discriminated at a rate of 10 Hz. We also examined the ability to detect the number of objects encapsulated in a droplet. Single cell droplets sorted into collection channels showed 91 ± 4.5% and 90 ± 3.8% accuracy for D. tertiolecta and P. tricornutum, respectively. Because we used image recognition to confirm single cell droplets, we achieved highly accurate single cell sorting. The results indicate that the integrated method of droplet imaging cell sorting can provide a complementary sorting approach capable of isolating single target cells from a mixture of cells with high accuracy without any staining.

An on-chip imaging droplet-sorting system: a real-time shape recognition method to screen target cells in droplets with single cell resolution.

PubMed

Girault, Mathias; Kim, Hyonchol; Arakawa, Hisayuki; Matsuura, Kenji; Odaka, Masao; Hattori, Akihiro; Terazono, Hideyuki; Yasuda, Kenji

2017-01-06

A microfluidic on-chip imaging cell sorter has several advantages over conventional cell sorting methods, especially to identify cells with complex morphologies such as clusters. One of the remaining problems is how to efficiently discriminate targets at the species level without labelling. Hence, we developed a label-free microfluidic droplet-sorting system based on image recognition of cells in droplets. To test the applicability of this method, a mixture of two plankton species with different morphologies (Dunaliella tertiolecta and Phaeodactylum tricornutum) were successfully identified and discriminated at a rate of 10 Hz. We also examined the ability to detect the number of objects encapsulated in a droplet. Single cell droplets sorted into collection channels showed 91 ± 4.5% and 90 ± 3.8% accuracy for D. tertiolecta and P. tricornutum, respectively. Because we used image recognition to confirm single cell droplets, we achieved highly accurate single cell sorting. The results indicate that the integrated method of droplet imaging cell sorting can provide a complementary sorting approach capable of isolating single target cells from a mixture of cells with high accuracy without any staining.

An on-chip imaging droplet-sorting system: a real-time shape recognition method to screen target cells in droplets with single cell resolution

PubMed Central

Girault, Mathias; Kim, Hyonchol; Arakawa, Hisayuki; Matsuura, Kenji; Odaka, Masao; Hattori, Akihiro; Terazono, Hideyuki; Yasuda, Kenji

2017-01-01

A microfluidic on-chip imaging cell sorter has several advantages over conventional cell sorting methods, especially to identify cells with complex morphologies such as clusters. One of the remaining problems is how to efficiently discriminate targets at the species level without labelling. Hence, we developed a label-free microfluidic droplet-sorting system based on image recognition of cells in droplets. To test the applicability of this method, a mixture of two plankton species with different morphologies (Dunaliella tertiolecta and Phaeodactylum tricornutum) were successfully identified and discriminated at a rate of 10 Hz. We also examined the ability to detect the number of objects encapsulated in a droplet. Single cell droplets sorted into collection channels showed 91 ± 4.5% and 90 ± 3.8% accuracy for D. tertiolecta and P. tricornutum, respectively. Because we used image recognition to confirm single cell droplets, we achieved highly accurate single cell sorting. The results indicate that the integrated method of droplet imaging cell sorting can provide a complementary sorting approach capable of isolating single target cells from a mixture of cells with high accuracy without any staining. PMID:28059147

Beat-to-Beat Variation in Periodicity of Local Calcium Releases Contributes to Intrinsic Variations of Spontaneous Cycle Length in Isolated Single Sinoatrial Node Cells

PubMed Central

Monfredi, Oliver; Maltseva, Larissa A.; Spurgeon, Harold A.; Boyett, Mark R.; Lakatta, Edward G.; Maltsev, Victor A.

2013-01-01

Spontaneous, submembrane local Ca2+ releases (LCRs) generated by the sarcoplasmic reticulum in sinoatrial nodal cells, the cells of the primary cardiac pacemaker, activate inward Na+/Ca2+-exchange current to accelerate the diastolic depolarization rate, and therefore to impact on cycle length. Since LCRs are generated by Ca2+ release channel (i.e. ryanodine receptor) openings, they exhibit a degree of stochastic behavior, manifested as notable cycle-to-cycle variations in the time of their occurrence. Aim The present study tested whether variation in LCR periodicity contributes to intrinsic (beat-to-beat) cycle length variability in single sinoatrial nodal cells. Methods We imaged single rabbit sinoatrial nodal cells using a 2D-camera to capture LCRs over the entire cell, and, in selected cells, simultaneously measured action potentials by perforated patch clamp. Results LCRs begin to occur on the descending part of the action potential-induced whole-cell Ca2+ transient, at about the time of the maximum diastolic potential. Shortly after the maximum diastolic potential (mean 54±7.7 ms, n = 14), the ensemble of waxing LCR activity converts the decay of the global Ca2+ transient into a rise, resulting in a late, whole-cell diastolic Ca2+ elevation, accompanied by a notable acceleration in diastolic depolarization rate. On average, cells (n = 9) generate 13.2±3.7 LCRs per cycle (mean±SEM), varying in size (7.1±4.2 µm) and duration (44.2±27.1 ms), with both size and duration being greater for later-occurring LCRs. While the timing of each LCR occurrence also varies, the LCR period (i.e. the time from the preceding Ca2+ transient peak to an LCR’s subsequent occurrence) averaged for all LCRs in a given cycle closely predicts the time of occurrence of the next action potential, i.e. the cycle length. Conclusion Intrinsic cycle length variability in single sinoatrial nodal cells is linked to beat-to-beat variations in the average period of individual LCRs each cycle. PMID:23826247

Hydrogel Droplet Microfluidics for High-Throughput Single Molecule/Cell Analysis.

PubMed

Zhu, Zhi; Yang, Chaoyong James

2017-01-17

Heterogeneity among individual molecules and cells has posed significant challenges to traditional bulk assays, due to the assumption of average behavior, which would lose important biological information in heterogeneity and result in a misleading interpretation. Single molecule/cell analysis has become an important and emerging field in biological and biomedical research for insights into heterogeneity between large populations at high resolution. Compared with the ensemble bulk method, single molecule/cell analysis explores the information on time trajectories, conformational states, and interactions of individual molecules/cells, all key factors in the study of chemical and biological reaction pathways. Various powerful techniques have been developed for single molecule/cell analysis, including flow cytometry, atomic force microscopy, optical and magnetic tweezers, single-molecule fluorescence spectroscopy, and so forth. However, some of them have the low-throughput issue that has to analyze single molecules/cells one by one. Flow cytometry is a widely used high-throughput technique for single cell analysis but lacks the ability for intercellular interaction study and local environment control. Droplet microfluidics becomes attractive for single molecule/cell manipulation because single molecules/cells can be individually encased in monodisperse microdroplets, allowing high-throughput analysis and manipulation with precise control of the local environment. Moreover, hydrogels, cross-linked polymer networks that swell in the presence of water, have been introduced into droplet microfluidic systems as hydrogel droplet microfluidics. By replacing an aqueous phase with a monomer or polymer solution, hydrogel droplets can be generated on microfluidic chips for encapsulation of single molecules/cells according to the Poisson distribution. The sol-gel transition property endows the hydrogel droplets with new functionalities and diversified applications in single molecule/cell analysis. The hydrogel can act as a 3D cell culture matrix to mimic the extracellular environment for long-term single cell culture, which allows further heterogeneity study in proliferation, drug screening, and metastasis at the single-cell level. The sol-gel transition allows reactions in solution to be performed rapidly and efficiently with product storage in the gel for flexible downstream manipulation and analysis. More importantly, controllable sol-gel regulation provides a new way to maintain phenotype-genotype linkages in the hydrogel matrix for high throughput molecular evolution. In this Account, we will review the hydrogel droplet generation on microfluidics, single molecule/cell encapsulation in hydrogel droplets, as well as the progress made by our group and others in the application of hydrogel droplet microfluidics for single molecule/cell analysis, including single cell culture, single molecule/cell detection, single cell sequencing, and molecular evolution.

Analyzing cell fate control by cytokines through continuous single cell biochemistry.

PubMed

Rieger, Michael A; Schroeder, Timm

2009-10-01

Cytokines are important regulators of cell fates with high clinical and commercial relevance. However, despite decades of intense academic and industrial research, it proved surprisingly difficult to describe the biological functions of cytokines in a precise and comprehensive manner. The exact analysis of cytokine biology is complicated by the fact that individual cytokines control many different cell fates and activate a multitude of intracellular signaling pathways. Moreover, although activating different molecular programs, different cytokines can be redundant in their biological effects. In addition, cytokines with different biological effects can activate overlapping signaling pathways. This prospect article will outline the necessity of continuous single cell biochemistry to unravel the biological functions of molecular cytokine signaling. It focuses on potentials and limitations of recent technical developments in fluorescent time-lapse imaging and single cell tracking allowing constant long-term observation of molecules and behavior of single cells. (c) 2009 Wiley-Liss, Inc.

Very Low Abundance Single-Cell Transcript Quantification with 5-Plex ddPCRTM Assays.

PubMed

Karlin-Neumann, George; Zhang, Bin; Litterst, Claudia

2018-01-01

Gene expression studies have provided one of the most accessible windows for understanding the molecular basis of cell and tissue phenotypes and how these change in response to stimuli. Current PCR-based and next generation sequencing methods offer great versatility in allowing the focused study of the roles of small numbers of genes or comprehensive profiling of the entire transcriptome of a sample at one time. Marrying of these approaches to various cell sorting technologies has recently enabled the profiling of expression in single cells, thereby increasing the resolution and sensitivity and strengthening the inferences from observed expression levels and changes. This chapter presents a quick and efficient 1-day workflow for sorting single cells with a small laboratory cell-sorter followed by an ultrahigh sensitivity, multiplexed digital PCR method for quantitative tracking of changes in 5-10 genes per single cell.

Single cell manipulation utilizing femtosecond laser-induced shock and stress waves

NASA Astrophysics Data System (ADS)

Hosokawa, Yoichiroh

2017-02-01

When an intense femtosecond laser pulse is focused into a culture medium through an objective lens, an impulsive force is loaded on the cells with generations of the shock and stress waves at the laser focal point. The shock and stress waves were acted to single cells in the vicinity of the laser focal point as an impulsive force. We have applied the impulsive force to manipulate single cells. As the transient intensity of the impulsive force is over 1000 times stronger than the force due to optical tweezers, drastic single manipulation which is difficult by the optical tweezers can be realized. The generation process of the impulsive force and behavior of animal cell after loading the impulsive force were reviewed, and then our original quantification method of the impulsive force utilizing atomic force microscope (AFM) was introduced with its applications for evaluating adhesions between animal cells and between sub-organelles in plant cell.

A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation.

PubMed

Nestorowa, Sonia; Hamey, Fiona K; Pijuan Sala, Blanca; Diamanti, Evangelia; Shepherd, Mairi; Laurenti, Elisa; Wilson, Nicola K; Kent, David G; Göttgens, Berthold

2016-08-25

Maintenance of the blood system requires balanced cell fate decisions by hematopoietic stem and progenitor cells (HSPCs). Because cell fate choices are executed at the individual cell level, new single-cell profiling technologies offer exciting possibilities for mapping the dynamic molecular changes underlying HSPC differentiation. Here, we have used single-cell RNA sequencing to profile more than 1600 single HSPCs, and deep sequencing has enabled detection of an average of 6558 protein-coding genes per cell. Index sorting, in combination with broad sorting gates, allowed us to retrospectively assign cells to 12 commonly sorted HSPC phenotypes while also capturing intermediate cells typically excluded by conventional gating. We further show that independently generated single-cell data sets can be projected onto the single-cell resolution expression map to directly compare data from multiple groups and to build and refine new hypotheses. Reconstruction of differentiation trajectories reveals dynamic expression changes associated with early lymphoid, erythroid, and granulocyte-macrophage differentiation. The latter two trajectories were characterized by common upregulation of cell cycle and oxidative phosphorylation transcriptional programs. By using external spike-in controls, we estimate absolute messenger RNA (mRNA) levels per cell, showing for the first time that despite a general reduction in total mRNA, a subset of genes shows higher expression levels in immature stem cells consistent with active maintenance of the stem-cell state. Finally, we report the development of an intuitive Web interface as a new community resource to permit visualization of gene expression in HSPCs at single-cell resolution for any gene of choice. © 2016 by The American Society of Hematology.

‘Living cantilever arrays’ for characterization of mass of single live cells in fluids†

PubMed Central

Park, Kidong; Jang, Jaesung; Irimia, Daniel; Sturgis, Jennifer; Lee, James; Robinson, J. Paul; Toner, Mehmet; Bashir, Rashid

2013-01-01

The size of a cell is a fundamental physiological property and is closely regulated by various environmental and genetic factors. Optical or confocal microscopy can be used to measure the dimensions of adherent cells, and Coulter counter or flow cytometry (forward scattering light intensity) can be used to estimate the volume of single cells in a flow. Although these methods could be used to obtain the mass of single live cells, no method suitable for directly measuring the mass of single adherent cells without detaching them from the surface is currently available. We report the design, fabrication, and testing of ‘living cantilever arrays’, an approach to measure the mass of single adherent live cells in fluid using silicon cantilever mass sensor. HeLa cells were injected into microfluidic channels with a linear array of functionalized silicon cantilevers and the cells were subsequently captured on the cantilevers with positive dielectrophoresis. The captured cells were then cultured on the cantilevers in a microfluidic environment and the resonant frequencies of the cantilevers were measured. The mass of a single HeLa cell was extracted from the resonance frequency shift of the cantilever and was found to be close to the mass value calculated from the cell density from the literature and the cell volume obtained from confocal microscopy. This approach can provide a new method for mass measurement of a single adherent cell in its physiological condition in a non-invasive manner, as well as optical observations of the same cell. We believe this technology would be very valuable for single cell time-course studies of adherent live cells. PMID:18584076

Mutation dynamics and fitness effects followed in single cells.

PubMed

Robert, Lydia; Ollion, Jean; Robert, Jerome; Song, Xiaohu; Matic, Ivan; Elez, Marina

2018-03-16

Mutations have been investigated for more than a century but remain difficult to observe directly in single cells, which limits the characterization of their dynamics and fitness effects. By combining microfluidics, time-lapse imaging, and a fluorescent tag of the mismatch repair system in Escherichia coli , we visualized the emergence of mutations in single cells, revealing Poissonian dynamics. Concomitantly, we tracked the growth and life span of single cells, accumulating ~20,000 mutations genome-wide over hundreds of generations. This analysis revealed that 1% of mutations were lethal; nonlethal mutations displayed a heavy-tailed distribution of fitness effects and were dominated by quasi-neutral mutations with an average cost of 0.3%. Our approach has enabled the investigation of single-cell individuality in mutation rate, mutation fitness costs, and mutation interactions. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Unexpected heterogeneity derived from Cas9 ribonucleoprotein-introduced clonal cells at the HPRT1 locus.

PubMed

Sakuma, Tetsushi; Mochida, Keiji; Nakade, Shota; Ezure, Toru; Minagawa, Sachi; Yamamoto, Takashi

2018-04-01

Single-cell cloning is an essential technique for establishing genome-edited cell clones mediated by programmable nucleases such as CRISPR-Cas9. However, residual genome-editing activity after single-cell cloning may cause heterogeneity in the clonal cells. Previous studies showed efficient mutagenesis and rapid degradation of CRISPR-Cas9 components in cultured cells by introducing Cas9 ribonucleoproteins (RNPs). In this study, we investigated how the timing for single-cell cloning of Cas9 RNP-transfected cells affected the heterogeneity of the resultant clones. We carried out transfection of Cas9 RNPs targeting several loci in the HPRT1 gene in HCT116 cells, followed by single-cell cloning at 24, 48, 72 hr and 1 week post-transfection. After approximately 3 weeks of incubation, the clonal cells were collected and genotyped by high-resolution microchip electrophoresis and Sanger sequencing. Unexpectedly, long-term incubation before single-cell cloning resulted in highly heterogeneous clones. We used a lipofection method for transfection, and the media containing transfectable RNPs were not removed before single-cell cloning. Therefore, the active Cas9 RNPs were considered to be continuously incorporated into cells during the precloning incubation. Our findings provide a warning that lipofection of Cas9 RNPs may cause continuous introduction of gene mutations depending on the experimental procedures. © 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Single-cell analysis by ICP-MS/MS as a fast tool for cellular bioavailability studies of arsenite.

PubMed

Meyer, S; López-Serrano, A; Mitze, H; Jakubowski, N; Schwerdtle, T

2018-01-24

Single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS) has become a powerful and fast tool to evaluate the elemental composition at a single-cell level. In this study, the cellular bioavailability of arsenite (incubation of 25 and 50 μM for 0-48 h) has been successfully assessed by SC-ICP-MS/MS for the first time directly after re-suspending the cells in water. This procedure avoids the normally arising cell membrane permeabilization caused by cell fixation methods (e.g. methanol fixation). The reliability and feasibility of this SC-ICP-MS/MS approach with a limit of detection of 0.35 fg per cell was validated by conventional bulk ICP-MS/MS analysis after cell digestion and parallel measurement of sulfur and phosphorus.

An open-pattern droplet-in-oil planar array for single cell analysis based on sequential inkjet printing technology.

PubMed

Wang, Chenyu; Liu, Wenwen; Tan, Manqing; Sun, Hongbo; Yu, Yude

2017-07-01

Cellular heterogeneity represents a fundamental principle of cell biology for which a readily available single-cell research tool is urgently required. Here, we present a novel method combining cell-sized well arrays with sequential inkjet printing. Briefly, K562 cells with phosphate buffer saline buffer were captured at high efficiency (74.5%) in a cell-sized well as a "primary droplet" and sealed using fluorinated oil. Then, piezoelectric inkjet printing technology was adapted to precisely inject the cell lysis buffer and the fluorogenic substrate, fluorescein-di-β-D-galactopyranoside, as a "secondary droplet" to penetrate the sealing oil and fuse with the "primary droplet." We thereby successfully measured the intracellular β-galactosidase activity of K562 cells at the single-cell level. Our method allows, for the first time, the ability to simultaneously accommodate the high occupancy rate of single cells and sequential addition of reagents while retaining an open structure. We believe that the feasibility and flexibility of our method will enhance its use as a universal single-cell research tool as well as accelerate the adoption of inkjet printing in the study of cellular heterogeneity.

Single-cell manipulation and DNA delivery technology using atomic force microscopy and nanoneedle.

PubMed

Han, Sung-Woong; Nakamura, Chikashi; Miyake, Jun; Chang, Sang-Mok; Adachi, Taiji

2014-01-01

The recent single-cell manipulation technology using atomic force microscopy (AFM) not only allows high-resolution visualization and probing of biomolecules and cells but also provides spatial and temporal access to the interior of living cells via the nanoneedle technology. Here we review the development and application of single-cell manipulations and the DNA delivery technology using a nanoneedle. We briefly describe various DNA delivery methods and discuss their advantages and disadvantages. Fabrication of the nanoneedle, visualization of nanoneedle insertion into living cells, DNA modification on the nanoneedle surface, and the invasiveness of nanoneedle insertion into living cells are described. Different methods of DNA delivery into a living cell, such as lipofection, microinjection, and nanoneedles, are then compared. Finally, single-cell diagnostics using the nanoneedle and the perspectives of the nanoneedle technology are outlined. The nanoneedle-based DNA delivery technology provides new opportunities for efficient and specific introduction of DNA and other biomolecules into precious living cells with a high spatial resolution within a desired time frame. This technology has the potential to be applied for many basic cellular studies and for clinical studies such as single-cell diagnostics.

Live single cell functional phenotyping in droplet nano-liter reactors.

PubMed

Konry, Tania; Golberg, Alexander; Yarmush, Martin

2013-11-11

While single cell heterogeneity is present in all biological systems, most studies cannot address it due to technical limitations. Here we describe a nano-liter droplet microfluidic-based approach for stimulation and monitoring of surface and secreted markers of live single immune dendritic cells (DCs) as well as monitoring the live T cell/DC interaction. This nano-liter in vivo simulating microenvironment allows delivering various stimuli reagents to each cell and appropriate gas exchanges which are necessary to ensure functionality and viability of encapsulated cells. Labeling bioassay and microsphere sensors were integrated into nano-liter reaction volume of the droplet to monitor live single cell surface markers and secretion analysis in the time-dependent fashion. Thus live cell stimulation, secretion and surface monitoring can be obtained simultaneously in distinct microenvironment, which previously was possible using complicated and multi-step in vitro and in vivo live-cell microscopy, together with immunological studies of the outcome secretion of cellular function.

SCNS: a graphical tool for reconstructing executable regulatory networks from single-cell genomic data.

PubMed

Woodhouse, Steven; Piterman, Nir; Wintersteiger, Christoph M; Göttgens, Berthold; Fisher, Jasmin

2018-05-25

Reconstruction of executable mechanistic models from single-cell gene expression data represents a powerful approach to understanding developmental and disease processes. New ambitious efforts like the Human Cell Atlas will soon lead to an explosion of data with potential for uncovering and understanding the regulatory networks which underlie the behaviour of all human cells. In order to take advantage of this data, however, there is a need for general-purpose, user-friendly and efficient computational tools that can be readily used by biologists who do not have specialist computer science knowledge. The Single Cell Network Synthesis toolkit (SCNS) is a general-purpose computational tool for the reconstruction and analysis of executable models from single-cell gene expression data. Through a graphical user interface, SCNS takes single-cell qPCR or RNA-sequencing data taken across a time course, and searches for logical rules that drive transitions from early cell states towards late cell states. Because the resulting reconstructed models are executable, they can be used to make predictions about the effect of specific gene perturbations on the generation of specific lineages. SCNS should be of broad interest to the growing number of researchers working in single-cell genomics and will help further facilitate the generation of valuable mechanistic insights into developmental, homeostatic and disease processes.

Analysis of population structures of the microalga Acutodesmus obliquus during lipid production using multi-dimensional single-cell analysis.

PubMed

Sandmann, Michael; Schafberg, Michaela; Lippold, Martin; Rohn, Sascha

2018-04-19

Microalgae bear a great potential to produce lipids for biodiesel, feed, or even food applications. To understand the still not well-known single-cell dynamics during lipid production in microalgae, a novel single-cell analytical technology was applied to study a well-established model experiment. Multidimensional single-cell dynamics were investigated with a non-supervised image analysis technique that utilizes data from epi-fluorescence microscopy. Reliability of this technique was successfully proven via reference analysis. The technique developed was used to determine cell size, chlorophyll amount, neutral lipid amount, and deriving properties on a single-cellular level in cultures of the biotechnologically promising alga Acutodesmus obliquus. The results illustrated a high correlation between cell size and chlorophyll amount, but a very low and dynamic correlation between cell size, lipid amount, and lipid density. During growth conditions under nitrogen starvation, cells with low chlorophyll content tend to start the lipid production first and the cell suspension differentiated in two subpopulations with significantly different lipid contents. Such quantitative characterization of single-cell dynamics of lipid synthesizing algae was done for the first time and the potential of such simple technology is highly relevant to other biotechnological applications and to deeper investigate the process of microalgal lipid accumulation.

Amplitude and timing properties of a Geiger discharge in a SiPM cell

NASA Astrophysics Data System (ADS)

Popova, E.; Buzhan, P.; Pleshko, A.; Vinogradov, S.; Stifutkin, A.; Ilyin, A.; Besson, D.; Mirzoyan, R.

2015-07-01

The amplitude and timing properties of a Geiger discharge in a stand-alone SiPM cell have been investigated in detail. Use of a single stand-alone SiPM cell allows us to perform measurements with better accuracy than the multicell structure of conventional SiPMs. We have studied the dependence of the output charge and amplitude from an SiPM cell illuminated by focused light vs the number of primary photoelectrons. We propose a SPICE model which explains the amplitude over saturation (when the SiPM's amplitude is greater than the sum over all cells) characteristics of SiPM signals for more than one initial photoelectrons. The time resolutions of a SiPM cell have been measured for the case of single (SPTR) and multiphoton light pulses. The Full Width Half Max (FWHM) for SPTR has been found to be at the level of 30 ps for focused and 40 ps for unfocused light (100 μm cell size).

Single cell transcriptomic analysis of prostate cancer cells.

PubMed

Welty, Christopher J; Coleman, Ilsa; Coleman, Roger; Lakely, Bryce; Xia, Jing; Chen, Shu; Gulati, Roman; Larson, Sandy R; Lange, Paul H; Montgomery, Bruce; Nelson, Peter S; Vessella, Robert L; Morrissey, Colm

2013-02-16

The ability to interrogate circulating tumor cells (CTC) and disseminated tumor cells (DTC) is restricted by the small number detected and isolated (typically <10). To determine if a commercially available technology could provide a transcriptomic profile of a single prostate cancer (PCa) cell, we clonally selected and cultured a single passage of cell cycle synchronized C4-2B PCa cells. Ten sets of single, 5-, or 10-cells were isolated using a micromanipulator under direct visualization with an inverted microscope. Additionally, two groups of 10 individual DTC, each isolated from bone marrow of 2 patients with metastatic PCa were obtained. RNA was amplified using the WT-Ovation™ One-Direct Amplification System. The amplified material was hybridized on a 44K Whole Human Gene Expression Microarray. A high stringency threshold, a mean Alexa Fluor® 3 signal intensity above 300, was used for gene detection. Relative expression levels were validated for select genes using real-time PCR (RT-qPCR). Using this approach, 22,410, 20,423, and 17,009 probes were positive on the arrays from 10-cell pools, 5-cell pools, and single-cells, respectively. The sensitivity and specificity of gene detection on the single-cell analyses were 0.739 and 0.972 respectively when compared to 10-cell pools, and 0.814 and 0.979 respectively when compared to 5-cell pools, demonstrating a low false positive rate. Among 10,000 randomly selected pairs of genes, the Pearson correlation coefficient was 0.875 between the single-cell and 5-cell pools and 0.783 between the single-cell and 10-cell pools. As expected, abundant transcripts in the 5- and 10-cell samples were detected by RT-qPCR in the single-cell isolates, while lower abundance messages were not. Using the same stringency, 16,039 probes were positive on the patient single-cell arrays. Cluster analysis showed that all 10 DTC grouped together within each patient. A transcriptomic profile can be reliably obtained from a single cell using commercially available technology. As expected, fewer amplified genes are detected from a single-cell sample than from pooled-cell samples, however this method can be used to reliably obtain a transcriptomic profile from DTC isolated from the bone marrow of patients with PCa.

Single-cell axotomy of cultured hippocampal neurons integrated in neuronal circuits.

PubMed

Gomis-Rüth, Susana; Stiess, Michael; Wierenga, Corette J; Meyn, Liane; Bradke, Frank

2014-05-01

An understanding of the molecular mechanisms of axon regeneration after injury is key for the development of potential therapies. Single-cell axotomy of dissociated neurons enables the study of the intrinsic regenerative capacities of injured axons. This protocol describes how to perform single-cell axotomy on dissociated hippocampal neurons containing synapses. Furthermore, to axotomize hippocampal neurons integrated in neuronal circuits, we describe how to set up coculture with a few fluorescently labeled neurons. This approach allows axotomy of single cells in a complex neuronal network and the observation of morphological and molecular changes during axon regeneration. Thus, single-cell axotomy of mature neurons is a valuable tool for gaining insights into cell intrinsic axon regeneration and the plasticity of neuronal polarity of mature neurons. Dissociation of the hippocampus and plating of hippocampal neurons takes ∼2 h. Neurons are then left to grow for 2 weeks, during which time they integrate into neuronal circuits. Subsequent axotomy takes 10 min per neuron and further imaging takes 10 min per neuron.

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations.

PubMed

Gómez-Villafuertes, Rosa; Paniagua-Herranz, Lucía; Gascon, Sergio; de Agustín-Durán, David; Ferreras, María de la O; Gil-Redondo, Juan Carlos; Queipo, María José; Menendez-Mendez, Aida; Pérez-Sen, Ráquel; Delicado, Esmerilda G; Gualix, Javier; Costa, Marcos R; Schroeder, Timm; Miras-Portugal, María Teresa; Ortega, Felipe

2017-12-16

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

cgCorrect: a method to correct for confounding cell-cell variation due to cell growth in single-cell transcriptomics

NASA Astrophysics Data System (ADS)

Blasi, Thomas; Buettner, Florian; Strasser, Michael K.; Marr, Carsten; Theis, Fabian J.

2017-06-01

Accessing gene expression at a single-cell level has unraveled often large heterogeneity among seemingly homogeneous cells, which remains obscured when using traditional population-based approaches. The computational analysis of single-cell transcriptomics data, however, still imposes unresolved challenges with respect to normalization, visualization and modeling the data. One such issue is differences in cell size, which introduce additional variability into the data and for which appropriate normalization techniques are needed. Otherwise, these differences in cell size may obscure genuine heterogeneities among cell populations and lead to overdispersed steady-state distributions of mRNA transcript numbers. We present cgCorrect, a statistical framework to correct for differences in cell size that are due to cell growth in single-cell transcriptomics data. We derive the probability for the cell-growth-corrected mRNA transcript number given the measured, cell size-dependent mRNA transcript number, based on the assumption that the average number of transcripts in a cell increases proportionally to the cell’s volume during the cell cycle. cgCorrect can be used for both data normalization and to analyze the steady-state distributions used to infer the gene expression mechanism. We demonstrate its applicability on both simulated data and single-cell quantitative real-time polymerase chain reaction (PCR) data from mouse blood stem and progenitor cells (and to quantitative single-cell RNA-sequencing data obtained from mouse embryonic stem cells). We show that correcting for differences in cell size affects the interpretation of the data obtained by typically performed computational analysis.

A study of pile-up in integrated time-correlated single photon counting systems

NASA Astrophysics Data System (ADS)

Arlt, Jochen; Tyndall, David; Rae, Bruce R.; Li, David D.-U.; Richardson, Justin A.; Henderson, Robert K.

2013-10-01

Recent demonstration of highly integrated, solid-state, time-correlated single photon counting (TCSPC) systems in CMOS technology is set to provide significant increases in performance over existing bulky, expensive hardware. Arrays of single photon single photon avalanche diode (SPAD) detectors, timing channels, and signal processing can be integrated on a single silicon chip with a degree of parallelism and computational speed that is unattainable by discrete photomultiplier tube and photon counting card solutions. New multi-channel, multi-detector TCSPC sensor architectures with greatly enhanced throughput due to minimal detector transit (dead) time or timing channel dead time are now feasible. In this paper, we study the potential for future integrated, solid-state TCSPC sensors to exceed the photon pile-up limit through analytic formula and simulation. The results are validated using a 10% fill factor SPAD array and an 8-channel, 52 ps resolution time-to-digital conversion architecture with embedded lifetime estimation. It is demonstrated that pile-up insensitive acquisition is attainable at greater than 10 times the pulse repetition rate providing over 60 dB of extended dynamic range to the TCSPC technique. Our results predict future CMOS TCSPC sensors capable of live-cell transient observations in confocal scanning microscopy, improved resolution of near-infrared optical tomography systems, and fluorescence lifetime activated cell sorting.

A study of pile-up in integrated time-correlated single photon counting systems.

PubMed

Arlt, Jochen; Tyndall, David; Rae, Bruce R; Li, David D-U; Richardson, Justin A; Henderson, Robert K

2013-10-01

Recent demonstration of highly integrated, solid-state, time-correlated single photon counting (TCSPC) systems in CMOS technology is set to provide significant increases in performance over existing bulky, expensive hardware. Arrays of single photon single photon avalanche diode (SPAD) detectors, timing channels, and signal processing can be integrated on a single silicon chip with a degree of parallelism and computational speed that is unattainable by discrete photomultiplier tube and photon counting card solutions. New multi-channel, multi-detector TCSPC sensor architectures with greatly enhanced throughput due to minimal detector transit (dead) time or timing channel dead time are now feasible. In this paper, we study the potential for future integrated, solid-state TCSPC sensors to exceed the photon pile-up limit through analytic formula and simulation. The results are validated using a 10% fill factor SPAD array and an 8-channel, 52 ps resolution time-to-digital conversion architecture with embedded lifetime estimation. It is demonstrated that pile-up insensitive acquisition is attainable at greater than 10 times the pulse repetition rate providing over 60 dB of extended dynamic range to the TCSPC technique. Our results predict future CMOS TCSPC sensors capable of live-cell transient observations in confocal scanning microscopy, improved resolution of near-infrared optical tomography systems, and fluorescence lifetime activated cell sorting.

Single-particle tracking of endocytosis and exocytosis of single-walled carbon nanotubes in NIH-3T3 cells.

PubMed

Jin, Hong; Heller, Daniel A; Strano, Michael S

2008-06-01

Over 10000 individual trajectories of nonphotobleaching single-walled carbon nanotubes (SWNT) were tracked as they are incorporated into and expelled from NIH-3T3 cells in real time on a perfusion microscope stage. An analysis of mean square displacement allows the complete construction of the mechanistic steps involved from single duration experiments. We observe the first conclusive evidence of SWNT exocytosis and show that the rate closely matches the endocytosis rate with negligible temporal offset. We identify and study the endocytosis and exocytosis pathway that leads to the previously observed aggregation and accumulation of SWNT within the cells.

In vivo imaging of T cell lymphoma infiltration process at the colon.

PubMed

Ueda, Yoshibumi; Ishiwata, Toshiyuki; Shinji, Seiichi; Arai, Tomio; Matsuda, Yoko; Aida, Junko; Sugimoto, Naotoshi; Okazaki, Toshiro; Kikuta, Junichi; Ishii, Masaru; Sato, Moritoshi

2018-03-05

The infiltration and proliferation of cancer cells in the secondary organs are of great interest, since they contribute to cancer metastasis. However, cancer cell dynamics in the secondary organs have not been elucidated at single-cell resolution. In the present study, we established an in vivo model using two-photon microscopy to observe how infiltrating cancer cells form assemblages from single T-cell lymphomas, EL4 cells, in the secondary organs. Using this model, after inoculation of EL4 cells in mice, we discovered that single EL4 cells infiltrated into the colon. In the early stage, sporadic elongated EL4 cells became lodged in small blood vessels. Real-time imaging revealed that, whereas more than 70% of EL4 cells did not move during a 1-hour observation, other EL4 cells irregularly moved even in small vessels and dynamically changed shape upon interacting with other cells. In the late stages, EL4 cells formed small nodules composed of several EL4 cells in blood vessels as well as crypts, suggesting the existence of diverse mechanisms of nodule formation. The present in vivo imaging system is instrumental to dissect cancer cell dynamics during metastasis in other organs at the single-cell level.

Optical computed tomography for spatially isotropic four-dimensional imaging of live single cells

PubMed Central

Kelbauskas, Laimonas; Shetty, Rishabh; Cao, Bin; Wang, Kuo-Chen; Smith, Dean; Wang, Hong; Chao, Shi-Hui; Gangaraju, Sandhya; Ashcroft, Brian; Kritzer, Margaret; Glenn, Honor; Johnson, Roger H.; Meldrum, Deirdre R.

2017-01-01

Quantitative three-dimensional (3D) computed tomography (CT) imaging of living single cells enables orientation-independent morphometric analysis of the intricacies of cellular physiology. Since its invention, x-ray CT has become indispensable in the clinic for diagnostic and prognostic purposes due to its quantitative absorption-based imaging in true 3D that allows objects of interest to be viewed and measured from any orientation. However, x-ray CT has not been useful at the level of single cells because there is insufficient contrast to form an image. Recently, optical CT has been developed successfully for fixed cells, but this technology called Cell-CT is incompatible with live-cell imaging due to the use of stains, such as hematoxylin, that are not compatible with cell viability. We present a novel development of optical CT for quantitative, multispectral functional 4D (three spatial + one spectral dimension) imaging of living single cells. The method applied to immune system cells offers truly isotropic 3D spatial resolution and enables time-resolved imaging studies of cells suspended in aqueous medium. Using live-cell optical CT, we found a heterogeneous response to mitochondrial fission inhibition in mouse macrophages and differential basal remodeling of small (0.1 to 1 fl) and large (1 to 20 fl) nuclear and mitochondrial structures on a 20- to 30-s time scale in human myelogenous leukemia cells. Because of its robust 3D measurement capabilities, live-cell optical CT represents a powerful new tool in the biomedical research field. PMID:29226240

Dynamics of lineage commitment revealed by single-cell transcriptomics of differentiating embryonic stem cells.

PubMed

Semrau, Stefan; Goldmann, Johanna E; Soumillon, Magali; Mikkelsen, Tarjei S; Jaenisch, Rudolf; van Oudenaarden, Alexander

2017-10-23

Gene expression heterogeneity in the pluripotent state of mouse embryonic stem cells (mESCs) has been increasingly well-characterized. In contrast, exit from pluripotency and lineage commitment have not been studied systematically at the single-cell level. Here we measure the gene expression dynamics of retinoic acid driven mESC differentiation from pluripotency to lineage commitment, using an unbiased single-cell transcriptomics approach. We find that the exit from pluripotency marks the start of a lineage transition as well as a transient phase of increased susceptibility to lineage specifying signals. Our study reveals several transcriptional signatures of this phase, including a sharp increase of gene expression variability and sequential expression of two classes of transcriptional regulators. In summary, we provide a comprehensive analysis of the exit from pluripotency and lineage commitment at the single cell level, a potential stepping stone to improved lineage manipulation through timing of differentiation cues.

iSBatch: a batch-processing platform for data analysis and exploration of live-cell single-molecule microscopy images and other hierarchical datasets.

PubMed

Caldas, Victor E A; Punter, Christiaan M; Ghodke, Harshad; Robinson, Andrew; van Oijen, Antoine M

2015-10-01

Recent technical advances have made it possible to visualize single molecules inside live cells. Microscopes with single-molecule sensitivity enable the imaging of low-abundance proteins, allowing for a quantitative characterization of molecular properties. Such data sets contain information on a wide spectrum of important molecular properties, with different aspects highlighted in different imaging strategies. The time-lapsed acquisition of images provides information on protein dynamics over long time scales, giving insight into expression dynamics and localization properties. Rapid burst imaging reveals properties of individual molecules in real-time, informing on their diffusion characteristics, binding dynamics and stoichiometries within complexes. This richness of information, however, adds significant complexity to analysis protocols. In general, large datasets of images must be collected and processed in order to produce statistically robust results and identify rare events. More importantly, as live-cell single-molecule measurements remain on the cutting edge of imaging, few protocols for analysis have been established and thus analysis strategies often need to be explored for each individual scenario. Existing analysis packages are geared towards either single-cell imaging data or in vitro single-molecule data and typically operate with highly specific algorithms developed for particular situations. Our tool, iSBatch, instead allows users to exploit the inherent flexibility of the popular open-source package ImageJ, providing a hierarchical framework in which existing plugins or custom macros may be executed over entire datasets or portions thereof. This strategy affords users freedom to explore new analysis protocols within large imaging datasets, while maintaining hierarchical relationships between experiments, samples, fields of view, cells, and individual molecules.

Single-molecule detection of protein efflux from microorganisms using fluorescent single-walled carbon nanotube sensor arrays

NASA Astrophysics Data System (ADS)

Landry, Markita Patricia; Ando, Hiroki; Chen, Allen Y.; Cao, Jicong; Kottadiel, Vishal Isaac; Chio, Linda; Yang, Darwin; Dong, Juyao; Lu, Timothy K.; Strano, Michael S.

2017-05-01

A distinct advantage of nanosensor arrays is their ability to achieve ultralow detection limits in solution by proximity placement to an analyte. Here, we demonstrate label-free detection of individual proteins from Escherichia coli (bacteria) and Pichia pastoris (yeast) immobilized in a microfluidic chamber, measuring protein efflux from single organisms in real time. The array is fabricated using non-covalent conjugation of an aptamer-anchor polynucleotide sequence to near-infrared emissive single-walled carbon nanotubes, using a variable chemical spacer shown to optimize sensor response. Unlabelled RAP1 GTPase and HIV integrase proteins were selectively detected from various cell lines, via large near-infrared fluorescent turn-on responses. We show that the process of E. coli induction, protein synthesis and protein export is highly stochastic, yielding variability in protein secretion, with E. coli cells undergoing division under starved conditions producing 66% fewer secreted protein products than their non-dividing counterparts. We further demonstrate the detection of a unique protein product resulting from T7 bacteriophage infection of E. coli, illustrating that nanosensor arrays can enable real-time, single-cell analysis of a broad range of protein products from various cell types.

Robust organelle size extractions from elastic scattering measurements of single cells (Conference Presentation)

NASA Astrophysics Data System (ADS)

Cannaday, Ashley E.; Draham, Robert; Berger, Andrew J.

2016-04-01

The goal of this project is to estimate non-nuclear organelle size distributions in single cells by measuring angular scattering patterns and fitting them with Mie theory. Simulations have indicated that the large relative size distribution of organelles (mean:width≈2) leads to unstable Mie fits unless scattering is collected at polar angles less than 20 degrees. Our optical system has therefore been modified to collect angles down to 10 degrees. Initial validations will be performed on polystyrene bead populations whose size distributions resemble those of cell organelles. Unlike with the narrow bead distributions that are often used for calibration, we expect to see an order-of-magnitude improvement in the stability of the size estimates as the minimum angle decreases from 20 to 10 degrees. Scattering patterns will then be acquired and analyzed from single cells (EMT6 mouse cancer cells), both fixed and live, at multiple time points. Fixed cells, with no changes in organelle sizes over time, will be measured to determine the fluctuation level in estimated size distribution due to measurement imperfections alone. Subsequent measurements on live cells will determine whether there is a higher level of fluctuation that could be attributed to dynamic changes in organelle size. Studies on unperturbed cells are precursors to ones in which the effects of exogenous agents are monitored over time.

Revealing the vectors of cellular identity with single-cell genomics

PubMed Central

Wagner, Allon; Regev, Aviv; Yosef, Nir

2017-01-01

Single-cell genomics has now made it possible to create a comprehensive atlas of human cells. At the same time, it has reopened definitions of a cell’s identity and type and of the ways in which they are regulated by the cell’s molecular circuitry. Emerging computational analysis methods, especially in single-cell RNA sequencing (scRNA-seq), have already begun to reveal, in a data-driven way, the diverse simultaneous facets of a cell’s identity, from a taxonomy of discrete cell types to continuous dynamic transitions and spatial locations. These developments will eventually allow a cell to be represented as a superposition of ‘basis vectors’, each determining a different (but possibly dependent) aspect of cellular organization and function. However, computational methods must also overcome considerable challenges—from handling technical noise and data scale to forming new abstractions of biology. As the scale of single-cell experiments continues to increase, new computational approaches will be essential for constructing and characterizing a reference map of cell identities. PMID:27824854

Fast measurement of proton exchange membrane fuel cell impedance based on pseudo-random binary sequence perturbation signals and continuous wavelet transform

NASA Astrophysics Data System (ADS)

Debenjak, Andrej; Boškoski, Pavle; Musizza, Bojan; Petrovčič, Janko; Juričić, Đani

2014-05-01

This paper proposes an approach to the estimation of PEM fuel cell impedance by utilizing pseudo-random binary sequence as a perturbation signal and continuous wavelet transform with Morlet mother wavelet. With the approach, the impedance characteristic in the frequency band from 0.1 Hz to 500 Hz is identified in 60 seconds, approximately five times faster compared to the conventional single-sine approach. The proposed approach was experimentally evaluated on a single PEM fuel cell of a larger fuel cell stack. The quality of the results remains at the same level compared to the single-sine approach.

Study of living single cells in culture: automated recognition of cell behavior.

PubMed

Bodin, P; Papin, S; Meyer, C; Travo, P

1988-07-01

An automated system capable of analyzing the behavior, in real time, of single living cells in culture, in a noninvasive and nondestructive way, has been developed. A large number of cell positions in single culture dishes were recorded using a computer controlled, robotized microscope. During subsequent observations, binary images obtained from video image analysis of the microscope visual field allowed the identification of the recorded cells. These cells could be revisited automatically every few minutes. Long-term studies of the behavior of cells make possible the analysis of cellular locomotary and mitotic activities as well as determination of cell shape (chosen from a defined library) for several hours or days in a fully automated way with observations spaced up to 30 minutes. Short-term studies of the behavior of cells permit the study, in a semiautomatic way, of acute effects of drugs (5 to 15 minutes) on changes of surface area and length of cells.

Toward a Droplet-Based Single-Cell Radiometric Assay.

PubMed

Gallina, Maria Elena; Kim, Tae Jin; Shelor, Mark; Vasquez, Jaime; Mongersun, Amy; Kim, Minkyu; Tang, Sindy K Y; Abbyad, Paul; Pratx, Guillem

2017-06-20

Radiotracers are widely used to track molecular processes, both in vitro and in vivo, with high sensitivity and specificity. However, most radionuclide detection methods have spatial resolution inadequate for single-cell analysis. A few existing methods can extract single-cell information from radioactive decays, but the stochastic nature of the process precludes high-throughput measurement (and sorting) of single cells. In this work, we introduce a new concept for translating radioactive decays occurring stochastically within radiolabeled single-cells into an integrated, long-lasting fluorescence signal. Single cells are encapsulated in radiofluorogenic droplets containing molecular probes sensitive to byproducts of ionizing radiation (primarily reactive oxygen species, or ROS). Different probes were examined in bulk solutions, and dihydrorhodamine 123 (DHRh 123) was selected as the lead candidate due to its sensitivity and reproducibility. Fluorescence intensity of DHRh 123 in bulk increased at a rate of 54% per Gy of X-ray radiation and 15% per MBq/ml of 2-deoxy-2-[ 18 F]-fluoro-d-glucose ([ 18 F]FDG). Fluorescence imaging of microfluidic droplets showed the same linear response, but droplets were less sensitive overall than the bulk ROS sensor (detection limit of 3 Gy per droplet). Finally, droplets encapsulating radiolabeled cancer cells allowed, for the first time, the detection of [ 18 F]FDG radiotracer uptake in single cells through fluorescence activation. With further improvements, we expect this technology to enable quantitative measurement and selective sorting of single cells based on the uptake of radiolabeled small molecules.

Optimization and evaluation of single-cell whole-genome multiple displacement amplification.

PubMed

Spits, C; Le Caignec, C; De Rycke, M; Van Haute, L; Van Steirteghem, A; Liebaers, I; Sermon, K

2006-05-01

The scarcity of genomic DNA can be a limiting factor in some fields of genetic research. One of the methods developed to overcome this difficulty is whole genome amplification (WGA). Recently, multiple displacement amplification (MDA) has proved very efficient in the WGA of small DNA samples and pools of cells, the reaction being catalyzed by the phi29 or the Bst DNA polymerases. The aim of the present study was to develop a reliable, efficient, and fast protocol for MDA at the single-cell level. We first compared the efficiency of phi29 and Bst polymerases on DNA samples and single cells. The phi29 polymerase generated accurately, in a short time and from a single cell, sufficient DNA for a large set of tests, whereas the Bst enzyme showed a low efficiency and a high error rate. A single-cell protocol was optimized using the phi29 polymerase and was evaluated on 60 single cells; the DNA obtained DNA was assessed by 22 locus-specific PCRs. This new protocol can be useful for many applications involving minute quantities of starting material, such as forensic DNA analysis, prenatal and preimplantation genetic diagnosis, or cancer research. (c) 2006 Wiley-Liss, Inc.

High-Throughput Accurate Single-Cell Screening of Euglena gracilis with Fluorescence-Assisted Optofluidic Time-Stretch Microscopy.

PubMed

Guo, Baoshan; Lei, Cheng; Ito, Takuro; Jiang, Yiyue; Ozeki, Yasuyuki; Goda, Keisuke

2016-01-01

The development of reliable, sustainable, and economical sources of alternative fuels is an important, but challenging goal for the world. As an alternative to liquid fossil fuels, algal biofuel is expected to play a key role in alleviating global warming since algae absorb atmospheric CO2 via photosynthesis. Among various algae for fuel production, Euglena gracilis is an attractive microalgal species as it is known to produce wax ester (good for biodiesel and aviation fuel) within lipid droplets. To date, while there exist many techniques for inducing microalgal cells to produce and accumulate lipid with high efficiency, few analytical methods are available for characterizing a population of such lipid-accumulated microalgae including E. gracilis with high throughout, high accuracy, and single-cell resolution simultaneously. Here we demonstrate high-throughput, high-accuracy, single-cell screening of E. gracilis with fluorescence-assisted optofluidic time-stretch microscopy-a method that combines the strengths of microfluidic cell focusing, optical time-stretch microscopy, and fluorescence detection used in conventional flow cytometry. Specifically, our fluorescence-assisted optofluidic time-stretch microscope consists of an optical time-stretch microscope and a fluorescence analyzer on top of a hydrodynamically focusing microfluidic device and can detect fluorescence from every E. gracilis cell in a population and simultaneously obtain its image with a high throughput of 10,000 cells/s. With the multi-dimensional information acquired by the system, we classify nitrogen-sufficient (ordinary) and nitrogen-deficient (lipid-accumulated) E. gracilis cells with a low false positive rate of 1.0%. This method holds promise for evaluating cultivation techniques and selective breeding for microalgae-based biofuel production.

Identification of innate lymphoid cells in single-cell RNA-Seq data.

PubMed

Suffiotti, Madeleine; Carmona, Santiago J; Jandus, Camilla; Gfeller, David

2017-07-01

Innate lymphoid cells (ILCs) consist of natural killer (NK) cells and non-cytotoxic ILCs that are broadly classified into ILC1, ILC2, and ILC3 subtypes. These cells recently emerged as important early effectors of innate immunity for their roles in tissue homeostasis and inflammation. Over the last few years, ILCs have been extensively studied in mouse and human at the functional and molecular level, including gene expression profiling. However, sorting ILCs with flow cytometry for gene expression analysis is a delicate and time-consuming process. Here we propose and validate a novel framework for studying ILCs at the transcriptomic level using single-cell RNA-Seq data. Our approach combines unsupervised clustering and a new cell type classifier trained on mouse ILC gene expression data. We show that this approach can accurately identify different ILCs, especially ILC2 cells, in human lymphocyte single-cell RNA-Seq data. Our new model relies only on genes conserved across vertebrates, thereby making it in principle applicable in any vertebrate species. Considering the rapid increase in throughput of single-cell RNA-Seq technology, our work provides a computational framework for studying ILC2 cells in single-cell transcriptomic data and may help exploring their conservation in distant vertebrate species.

Automated assembling of single fuel cell units for use in a fuel cell stack

NASA Astrophysics Data System (ADS)

Jalba, C. K.; Muminovic, A.; Barz, C.; Nasui, V.

2017-05-01

The manufacturing of PEMFC stacks (POLYMER ELEKTROLYT MEMBRAN Fuel Cell) is nowadays still done by hand. Over hundreds of identical single components have to be placed accurate together for the construction of a fuel cell stack. Beside logistic problems, higher total costs and disadvantages in weight the high number of components produce a higher statistic interference because of faulty erection or material defects and summation of manufacturing tolerances. The saving of costs is about 20 - 25 %. Furthermore, the total weight of the fuel cells will be reduced because of a new sealing technology. Overall a one minute cycle time has to be aimed per cell at the manufacturing of these single components. The change of the existing sealing concept to a bonded sealing is one of the important requisites to get an automated manufacturing of single cell units. One of the important steps for an automated gluing process is the checking of the glue application by using of an image processing system. After bonding the single fuel cell the sealing and electrical function can be checked, so that only functional and high qualitative cells can get into further manufacturing processes.

Optimization of a therapeutic protocol for intravenous injection of human mesenchymal stem cells after cerebral ischemia in adult rats.

PubMed

Omori, Yoshinori; Honmou, Osamu; Harada, Kuniaki; Suzuki, Junpei; Houkin, Kiyohiro; Kocsis, Jeffery D

2008-10-21

The systemic injection of human mesenchymal stem cells (hMSCs) prepared from adult bone marrow has therapeutic benefits after cerebral artery occlusion in rats, and may have multiple therapeutic effects at various sites and times within the lesion as the cells respond to a particular pathological microenvironment. However, the comparative therapeutic benefits of multiple injections of hMSCs at different time points after cerebral artery occlusion in rats remain unclear. In this study, we induced middle cerebral artery occlusion (MCAO) in rats using intra-luminal vascular occlusion, and infused hMSCs intravenously at a single 6 h time point (low and high cell doses) and various multiple time points after MCAO. From MRI analyses lesion volume was reduced in all hMSC cell injection groups as compared to serum alone injections. However, the greatest therapeutic benefit was achieved following a single high cell dose injection at 6 h post-MCAO, rather than multiple lower cell infusions over multiple time points. Three-dimensional analysis of capillary vessels in the lesion indicated that the capillary volume was equally increased in all of the cell-injected groups. Thus, differences in functional outcome in the hMSC transplantation subgroups are not likely the result of differences in angiogenesis, but rather from differences in neuroprotective effects.

Properties of Single K+ and Cl− Channels in Asclepias tuberosa Protoplasts 1

PubMed Central

Schauf, Charles L.; Wilson, Kathryn J.

1987-01-01

Potassium and chloride channels were characterized in Asclepias tuberosa suspension cell derived protoplasts by patch voltage-clamp. Whole-cell currents and single channels in excised patches had linear instantaneous current-voltage relations, reversing at the Nernst potentials for K+ and Cl−, respectively. Whole cell K+ currents activated exponentially during step depolarizations, while voltage-dependent Cl− channels were activated by hyperpolarizations. Single K+ channel conductance was 40 ± 5 pS with a mean open time of 4.5 milliseconds at 100 millivolts. Potassium channels were blocked by Cs+ and tetraethylammonium, but were insensitive to 4-aminopyridine. Chloride channels had a single-channel conductance of 100 ± 17 picosiemens, mean open time of 8.8 milliseconds, and were blocked by Zn2+ and ethacrynic acid. Whole-cell Cl− currents were inhibited by abscisic acid, and were unaffected by indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid. Since internal and external composition can be controlled, patch-clamped protoplasts are ideal systems for studying the role of ion channels in plant physiology and development. Images Fig. 5 PMID:16665712

Microfluidic platform for single cell analysis under dynamic spatial and temporal stimulation.

PubMed

Song, Jiyoung; Ryu, Hyunryul; Chung, Minhwan; Kim, Youngtaek; Blum, Yannick; Lee, Sung Sik; Pertz, Olivier; Jeon, Noo Li

2018-05-01

Recent research on cellular responses is shifting from static observations recorded under static stimuli to real-time monitoring in a dynamic environment. Since cells sense and interact with their surrounding microenvironment, an experimental platform where dynamically changing cellular microenvironments should be recreated in vitro. There has been a lack of microfluidic devices to support spatial and temporal stimulations in a simple and robust manner. Here, we describe a microfluidic device that generates dynamic chemical gradients and pulses in both space and time using a single device. This microfluidic device provides at least 12h of continuous stimulations that can be used to observe responses from mammalian cells. Combination of the microfluidic de-vice with live-cell imaging facilitates real-time observation of dynamic cellular response at single cell level. Using stable HEK cells with biosensors, ERK (Extracellular signal-Regulated Kinase) activities were observed un-der the pulsatile and ramping stimulations of EGF (Epidermal Growth Factor). We quantified ERK activation even at extremely low EGF concentration (0.0625µg/ml), which can not be observed using conventional techniques such as western blot. Cytoskeleton re-arrangement of the 3T3 fibroblast (stable transfection with Lifeact-GFP) was compared under abrupt and gradually changing gradient of PDGF. Copyright © 2017 Elsevier B.V. All rights reserved.

Separation and parallel sequencing of the genomes and transcriptomes of single cells using G&T-seq.

PubMed

Macaulay, Iain C; Teng, Mabel J; Haerty, Wilfried; Kumar, Parveen; Ponting, Chris P; Voet, Thierry

2016-11-01

Parallel sequencing of a single cell's genome and transcriptome provides a powerful tool for dissecting genetic variation and its relationship with gene expression. Here we present a detailed protocol for G&T-seq, a method for separation and parallel sequencing of genomic DNA and full-length polyA(+) mRNA from single cells. We provide step-by-step instructions for the isolation and lysis of single cells; the physical separation of polyA(+) mRNA from genomic DNA using a modified oligo-dT bead capture and the respective whole-transcriptome and whole-genome amplifications; and library preparation and sequence analyses of these amplification products. The method allows the detection of thousands of transcripts in parallel with the genetic variants captured by the DNA-seq data from the same single cell. G&T-seq differs from other currently available methods for parallel DNA and RNA sequencing from single cells, as it involves physical separation of the DNA and RNA and does not require bespoke microfluidics platforms. The process can be implemented manually or through automation. When performed manually, paired genome and transcriptome sequencing libraries from eight single cells can be produced in ∼3 d by researchers experienced in molecular laboratory work. For users with experience in the programming and operation of liquid-handling robots, paired DNA and RNA libraries from 96 single cells can be produced in the same time frame. Sequence analysis and integration of single-cell G&T-seq DNA and RNA data requires a high level of bioinformatics expertise and familiarity with a wide range of informatics tools.

Elastic light scattering from single cells: orientational dynamics in optical trap.

PubMed

Watson, Dakota; Hagen, Norbert; Diver, Jonathan; Marchand, Philippe; Chachisvilis, Mirianas

2004-08-01

Light-scattering diagrams (phase functions) from single living cells and beads suspended in an optical trap were recorded with 30-ms time resolution. The intensity of the scattered light was recorded over an angular range of 0.5-179.5 degrees using an optical setup based on an elliptical mirror and rotating aperture. Experiments revealed that light-scattering diagrams from biological cells exhibit significant and complex time dependence. We have attributed this dependence to the cell's orientational dynamics within the trap. We have also used experimentally measured phase function information to calculate the time dependence of the optical radiation pressure force on the trapped particle and show how it changes depending on the orientation of the particle. Relevance of these experiments to potential improvement in the sensitivity of label-free flow cytometry is discussed.

Single Cell Mathematical Model Successfully Replicates Key Features of GBM: Go-Or-Grow Is Not Necessary.

PubMed

Scribner, Elizabeth; Fathallah-Shaykh, Hassan M

2017-01-01

Glioblastoma (GBM) is a malignant brain tumor that continues to be associated with neurological morbidity and poor survival times. Brain invasion is a fundamental property of malignant glioma cells. The Go-or-Grow (GoG) phenotype proposes that cancer cell motility and proliferation are mutually exclusive. Here, we construct and apply a single glioma cell mathematical model that includes motility and angiogenesis and lacks the GoG phenotype. Simulations replicate key features of GBM including its multilayer structure (i.e.edema, enhancement, and necrosis), its progression patterns associated with bevacizumab treatment, and replicate the survival times of GBM treated or untreated with bevacizumab. These results suggest that the GoG phenotype is not a necessary property for the formation of the multilayer structure, recurrence patterns, and the poor survival times of patients diagnosed with GBM.

Micromagnetic Cancer Cell Immobilization and Release for Real-Time Single Cell Analysis

NASA Astrophysics Data System (ADS)

Jaiswal, Devina; Rad, Armin Tahmasbi; Nieh, Mu-Ping; Claffey, Kevin P.; Hoshino, Kazunori

2017-04-01

Understanding the interaction of live cells with macromolecules is crucial for designing efficient therapies. Considering the functional heterogeneity found in cancer cells, real-time single cell analysis is necessary to characterize responses. In this study, we have designed and fabricated a microfluidic channel with patterned micromagnets which can temporarily immobilize the cells during analysis and release them after measurements. The microchannel is composed of plain coverslip top and bottom panels to facilitate easy microscopic observation and undisturbed application of analytes to the cells. Cells labeled with functionalized magnetic beads were immobilized in the device with an efficiency of 90.8±3.6%. Since the micromagnets are made of soft magnetic material (Ni), they released cells when external magnetic field was turned off from the channel. This allows the reuse of the channel for a new sample. As a model drug analysis, the immobilized breast cancer cells (MCF7) were exposed to fluorescent lipid nanoparticles and association and dissociation were measured through fluorescence analysis. Two concentrations of nanoparticles, 0.06 μg/ml and 0.08 μg/ml were tested and time lapse images were recorded and analyzed. The microfluidic device was able to provide a microenvironment for sample analysis, making it an efficient platform for real-time analysis.

Magnetic fingerprints of rolling cells for quantitative flow cytometry in whole blood

NASA Astrophysics Data System (ADS)

Reisbeck, Mathias; Helou, Michael Johannes; Richter, Lukas; Kappes, Barbara; Friedrich, Oliver; Hayden, Oliver

2016-09-01

Over the past 50 years, flow cytometry has had a profound impact on preclinical and clinical applications requiring single cell function information for counting, sub-typing and quantification of epitope expression. At the same time, the workflow complexity and high costs of such optical systems still limit flow cytometry applications to specialized laboratories. Here, we present a quantitative magnetic flow cytometer that incorporates in situ magnetophoretic cell focusing for highly accurate and reproducible rolling of the cellular targets over giant magnetoresistance sensing elements. Time-of-flight analysis is used to unveil quantitative single cell information contained in its magnetic fingerprint. Furthermore, we used erythrocytes as a biological model to validate our methodology with respect to precise analysis of the hydrodynamic cell diameter, quantification of binding capacity of immunomagnetic labels, and discrimination of cell morphology. The extracted time-of-flight information should enable point-of-care quantitative flow cytometry in whole blood for clinical applications, such as immunology and primary hemostasis.

Method for physiologic phenotype characterization at the single-cell level in non-interacting and interacting cells

PubMed Central

Ashili, Shashanka P.; Houkal, Jeff; Smith, Dean; Mohammadreza, Aida; Lee, Kristen B.; Forrester, Jessica; Kumar, Ashok; Anis, Yasser H.; Paulson, Thomas G.; Youngbull, Cody A.; Tian, Yanqing; Holl, Mark R.; Johnson, Roger H.; Meldrum, Deirdre R.

2012-01-01

Abstract. Intercellular heterogeneity is a key factor in a variety of core cellular processes including proliferation, stimulus response, carcinogenesis, and drug resistance. However, cell-to-cell variability studies at the single-cell level have been hampered by the lack of enabling experimental techniques. We present a measurement platform that features the capability to quantify oxygen consumption rates of individual, non-interacting and interacting cells under normoxic and hypoxic conditions. It is based on real-time concentration measurements of metabolites of interest by means of extracellular optical sensors in cell-isolating microwells of subnanoliter volume. We present the results of a series of measurements of oxygen consumption rates (OCRs) of individual non-interacting and interacting human epithelial cells. We measured the effects of cell-to-cell interactions by using the system’s capability to isolate two and three cells in a single well. The major advantages of the approach are: 1. ratiometric, intensity-based characterization of the metabolic phenotype at the single-cell level, 2. minimal invasiveness due to the distant positioning of sensors, and 3. ability to study the effects of cell-cell interactions on cellular respiration rates. PMID:22502580

Method for physiologic phenotype characterization at the single-cell level in non-interacting and interacting cells.

PubMed

Kelbauskas, Laimonas; Ashili, Shashanka P; Houkal, Jeff; Smith, Dean; Mohammadreza, Aida; Lee, Kristen B; Forrester, Jessica; Kumar, Ashok; Anis, Yasser H; Paulson, Thomas G; Youngbull, Cody A; Tian, Yanqing; Holl, Mark R; Johnson, Roger H; Meldrum, Deirdre R

2012-03-01

Intercellular heterogeneity is a key factor in a variety of core cellular processes including proliferation, stimulus response, carcinogenesis, and drug resistance. However, cell-to-cell variability studies at the single-cell level have been hampered by the lack of enabling experimental techniques. We present a measurement platform that features the capability to quantify oxygen consumption rates of individual, non-interacting and interacting cells under normoxic and hypoxic conditions. It is based on real-time concentration measurements of metabolites of interest by means of extracellular optical sensors in cell-isolating microwells of subnanoliter volume. We present the results of a series of measurements of oxygen consumption rates (OCRs) of individual non-interacting and interacting human epithelial cells. We measured the effects of cell-to-cell interactions by using the system's capability to isolate two and three cells in a single well. The major advantages of the approach are: 1. ratiometric, intensity-based characterization of the metabolic phenotype at the single-cell level, 2. minimal invasiveness due to the distant positioning of sensors, and 3. ability to study the effects of cell-cell interactions on cellular respiration rates. © 2012 Society of Photo-Optical Instrumentation Engineers (SPIE).

Making sense of snapshot data: ergodic principle for clonal cell populations

PubMed Central

2017-01-01

Population growth is often ignored when quantifying gene expression levels across clonal cell populations. We develop a framework for obtaining the molecule number distributions in an exponentially growing cell population taking into account its age structure. In the presence of generation time variability, the average acquired across a population snapshot does not obey the average of a dividing cell over time, apparently contradicting ergodicity between single cells and the population. Instead, we show that the variation observed across snapshots with known cell age is captured by cell histories, a single-cell measure obtained from tracking an arbitrary cell of the population back to the ancestor from which it originated. The correspondence between cells of known age in a population with their histories represents an ergodic principle that provides a new interpretation of population snapshot data. We illustrate the principle using analytical solutions of stochastic gene expression models in cell populations with arbitrary generation time distributions. We further elucidate that the principle breaks down for biochemical reactions that are under selection, such as the expression of genes conveying antibiotic resistance, which gives rise to an experimental criterion with which to probe selection on gene expression fluctuations. PMID:29187636

Making sense of snapshot data: ergodic principle for clonal cell populations.

PubMed

Thomas, Philipp

2017-11-01

Population growth is often ignored when quantifying gene expression levels across clonal cell populations. We develop a framework for obtaining the molecule number distributions in an exponentially growing cell population taking into account its age structure. In the presence of generation time variability, the average acquired across a population snapshot does not obey the average of a dividing cell over time, apparently contradicting ergodicity between single cells and the population. Instead, we show that the variation observed across snapshots with known cell age is captured by cell histories, a single-cell measure obtained from tracking an arbitrary cell of the population back to the ancestor from which it originated. The correspondence between cells of known age in a population with their histories represents an ergodic principle that provides a new interpretation of population snapshot data. We illustrate the principle using analytical solutions of stochastic gene expression models in cell populations with arbitrary generation time distributions. We further elucidate that the principle breaks down for biochemical reactions that are under selection, such as the expression of genes conveying antibiotic resistance, which gives rise to an experimental criterion with which to probe selection on gene expression fluctuations. © 2017 The Author(s).

Effects of time-variant extremely low-frequency (ELF) electromagnetic fields (EMF) on cholinesterase activity in Dictyostelium discoideum (Protista).

PubMed

Amaroli, Andrea; Trielli, Francesca; Bianco, Bruno; Giordano, Stefano; Moggia, Elsa; Corrado, Maria U Delmonte

2005-12-15

Recently, we detected propionylcholinesterase (PrChE) activity in single-cell amoebae of Dictyostelium discoideum using cytochemical, electrophoretic, and spectrophotometric methods. The involvement of this enzyme activity in cell-cell and cell-environment interactions was suggested. In this work, we found that exposure of single-cell amoebae to an extremely low-frequency electromagnetic fields (ELF-EMF) of 300 microT, 50 Hz, from 1 h up to 48 h at 21 +/- 1 degrees C affected PrChE activity.

Dynamic quantitative analysis of adherent cell cultures by means of lens-free video microscopy

NASA Astrophysics Data System (ADS)

Allier, C.; Vincent, R.; Navarro, F.; Menneteau, M.; Ghenim, L.; Gidrol, X.; Bordy, T.; Hervé, L.; Cioni, O.; Bardin, S.; Bornens, M.; Usson, Y.; Morales, S.

2018-02-01

We present our implementation of lens-free video microscopy setup for the monitoring of adherent cell cultures. We use a multi-wavelength LED illumination together with a dedicated holographic reconstruction algorithm that allows for an efficient removal of twin images from the reconstructed phase image for densities up to those of confluent cell cultures (>500 cells/mm2). We thereby demonstrate that lens-free video microscopy, with a large field of view ( 30 mm2) can enable us to capture the images of thousands of cells simultaneously and directly inside the incubator. It is then possible to trace and quantify single cells along several cell cycles. We thus prove that lens-free microscopy is a quantitative phase imaging technique enabling estimation of several metrics at the single cell level as a function of time, for example the area, dry mass, maximum thickness, major axis length and aspect ratio of each cell. Combined with cell tracking, it is then possible to extract important parameters such as the initial cell dry mass (just after cell division), the final cell dry mass (just before cell division), the average cell growth rate, and the cell cycle duration. As an example, we discuss the monitoring of a HeLa cell cultures which provided us with a data-set featuring more than 10 000 cell cycle tracks and more than 2x106 cell morphological measurements in a single time-lapse.

Multiple displacement amplification on single cell and possible PGD applications.

PubMed

Hellani, Ali; Coskun, Serdar; Benkhalifa, Moncef; Tbakhi, Abelghani; Sakati, Nadia; Al-Odaib, Ali; Ozand, Pinar

2004-11-01

Multiple displacement amplification (MDA) is a technique used in the amplification of very low amounts of DNA and reported to yield large quantities of high-quality DNA. We used MDA to amplify the whole genome directly from a single cell. The most common techniques used in PGD are PCR and fluorescent in-situ hybridization (FISH). There are many limitations to these techniques including, the number of chromosomes diagnosed for FISH or the quality of DNA issued from a single cell PCR. This report shows, for the first time, use of MDA for single cell whole genome amplification. A total of 16 short tandem repeats (STRs) were amplified successfully with a similar pattern to the genomic DNA. Furthermore, allelic drop out (ADO) derived from MDA was assessed in 40 single cells by analysing (i) heterozygosity for a known beta globin mutation (IVSI-5 C-G) and by studying (ii) the heterozygous loci present in the STRs. ADO turned out to be 10.25% for the beta globin gene sequencing and 5% for the fluorescent PCR analysis of STRs. Moreover, the amplification accuracy of MDA permitted the detection of trisomy 21 on a single cell using comparative genome hybridization-array. Altogether, these data suggest that MDA can be used for single cell molecular karyotyping and the diagnosis of any single gene disorder in PGD.

Single-cell and subcellular pharmacokinetic imaging allows insight into drug action in vivo.

PubMed

Thurber, Greg M; Yang, Katy S; Reiner, Thomas; Kohler, Rainer H; Sorger, Peter; Mitchison, Tim; Weissleder, Ralph

2013-01-01

Pharmacokinetic analysis at the organ level provides insight into how drugs distribute throughout the body, but cannot explain how drugs work at the cellular level. Here we demonstrate in vivo single-cell pharmacokinetic imaging of PARP-1 inhibitors and model drug behaviour under varying conditions. We visualize intracellular kinetics of the PARP-1 inhibitor distribution in real time, showing that PARP-1 inhibitors reach their cellular target compartment, the nucleus, within minutes in vivo both in cancer and normal cells in various cancer models. We also use these data to validate predictive finite element modelling. Our theoretical and experimental data indicate that tumour cells are exposed to sufficiently high PARP-1 inhibitor concentrations in vivo and suggest that drug inefficiency is likely related to proteomic heterogeneity or insensitivity of cancer cells to DNA-repair inhibition. This suggests that single-cell pharmacokinetic imaging and derived modelling improve our understanding of drug action at single-cell resolution in vivo.

Exploring dynamics in living cells by tracking single particles.

PubMed

Levi, Valeria; Gratton, Enrico

2007-01-01

In the last years, significant advances in microscopy techniques and the introduction of a novel technology to label living cells with genetically encoded fluorescent proteins revolutionized the field of Cell Biology. Our understanding on cell dynamics built from snapshots on fixed specimens has evolved thanks to our actual capability to monitor in real time the evolution of processes in living cells. Among these new tools, single particle tracking techniques were developed to observe and follow individual particles. Hence, we are starting to unravel the mechanisms driving the motion of a wide variety of cellular components ranging from organelles to protein molecules by following their way through the cell. In this review, we introduce the single particle tracking technology to new users. We briefly describe the instrumentation and explain some of the algorithms commonly used to locate and track particles. Also, we present some common tools used to analyze trajectories and illustrate with some examples the applications of single particle tracking to study dynamics in living cells.

Embryonic stem cells improve skeletal muscle recovery after extreme atrophy in mice.

PubMed

Artioli, Guilherme Giannini; De Oliveira Silvestre, João Guilherme; Guilherme, João Paulo Limongi França; Baptista, Igor Luchini; Ramos, Gracielle Vieira; Da Silva, Willian José; Miyabara, Elen Haruka; Moriscot, Anselmo Sigari

2015-03-01

We injected embryonic stem cells into mouse tibialis anterior muscles subjected to botulinum toxin injections as a model for reversible neurogenic atrophy. Muscles were exposed to botulinum toxin for 4 weeks and allowed to recover for up to 6 weeks. At the onset of recovery, a single muscle injection of embryonic stem cells was administered. The myofiber cross-sectional area, single twitch force, peak tetanic force, time-to-peak force, and half-relaxation time were determined. Although the stem cell injection did not affect the myofiber cross-sectional area gain in recovering muscles, most functional parameters improved significantly compared with those of recovering muscles that did not receive the stem cell injection. Muscle function recovery was accelerated by embryonic stem cell delivery in this durable neurogenic atrophy model. We conclude that stem cells should be considered a potential therapeutic tool for recovery after extreme skeletal muscle atrophy. © 2014 Wiley Periodicals, Inc.

Microfluidic platform for real-time signaling analysis of multiple single T cells in parallel.

PubMed

Faley, Shannon; Seale, Kevin; Hughey, Jacob; Schaffer, David K; VanCompernolle, Scott; McKinney, Brett; Baudenbacher, Franz; Unutmaz, Derya; Wikswo, John P

2008-10-01

Deciphering the signaling pathways that govern stimulation of naïve CD4+ T helper cells by antigen-presenting cells via formation of the immunological synapse is key to a fundamental understanding of the progression of successful adaptive immune response. The study of T cell-APC interactions in vitro is challenging, however, due to the difficulty of tracking individual, non-adherent cell pairs over time. Studying single cell dynamics over time reveals rare, but critical, signaling events that might be averaged out in bulk experiments, but these less common events are undoubtedly important for an integrated understanding of a cellular response to its microenvironment. We describe a novel application of microfluidic technology that overcomes many limitations of conventional cell culture and enables the study of hundreds of passively sequestered hematopoietic cells for extended periods of time. This microfluidic cell trap device consists of 440 18 micromx18 micromx10 microm PDMS, bucket-like structures opposing the direction of flow which serve as corrals for cells as they pass through the cell trap region. Cell viability analysis revealed that more than 70% of naïve CD4+ T cells (TN), held in place using only hydrodynamic forces, subsequently remain viable for 24 hours. Cytosolic calcium transients were successfully induced in TN cells following introduction of chemical, antibody, or cellular forms of stimulation. Statistical analysis of TN cells from a single stimulation experiment reveals the power of this platform to distinguish different calcium response patterns, an ability that might be utilized to characterize T cell signaling states in a given population. Finally, we investigate in real time contact- and non-contact-based interactions between primary T cells and dendritic cells, two main participants in the formation of the immunological synapse. Utilizing the microfluidic traps in a daisy-chain configuration allowed us to observe calcium transients in TN cells exposed only to media conditioned by secretions of lipopolysaccharide-matured dendritic cells, an event which is easily missed in conventional cell culture where large media-to-cell ratios dilute cellular products. Further investigation into this intercellular signaling event indicated that LPS-matured dendritic cells, in the absence of antigenic stimulation, secrete chemical signals that induce calcium transients in T(N) cells. While the stimulating factor(s) produced by the mature dendritic cells remains to be identified, this report illustrates the utility of these microfluidic cell traps for analyzing arrays of individual suspension cells over time and probing both contact-based and intercellular signaling events between one or more cell populations.

Effects of fotemustine or dacarbasine on a melanoma cell line pretreated with therapeutic proton irradiation

PubMed Central

Ristić-Fira, Aleksandra M; Korićanac, Lela B; Žakula, Jelena J; Valastro, Lucia M; Iannolo, Gioacchin; Privitera, Giuseppe; Cuttone, Giacomo; Petrović, Ivan M

2009-01-01

Background Considering that HTB140 melanoma cells have shown a poor response to either protons or alkylating agents, the effects of a combined use of these agents have been analysed. Methods Cells were irradiated in the middle of the therapeutic 62 MeV proton spread out Bragg peak (SOBP). Irradiation doses were 12 or 16 Gy and are those frequently used in proton therapy. Four days after irradiation cells were treated with fotemustine (FM) or dacarbazine (DTIC). Drug concentrations were 100 and 250 μM, values close to those that produce 50% of growth inhibition. Cell viability, proliferation, survival and cell cycle distribution were assessed 7 days after irradiation that corresponds to more than six doubling times of HTB140 cells. In this way incubation periods providing the best single effects of drugs (3 days) and protons (7 days) coincided at the same time. Results Single proton irradiations have reduced the number of cells to ~50%. FM caused stronger cell inactivation due to its high toxicity, while the effectiveness of DTIC, that was important at short term, almost vanished with the incubation of 7 days. Cellular mechanisms triggered by proton irradiation differently influenced the final effects of combined treatments. Combination of protons and FM did not improve cell inactivation level achieved by single treatments. A low efficiency of the single DTIC treatment was overcome when DTIC was introduced following proton irradiation, giving better inhibitory effects with respect to the single treatments. Most of the analysed cells were in G1/S phase, viable, active and able to replicate DNA. Conclusion The obtained results are the consequence of a high resistance of HTB140 melanoma cells to protons and/or drugs. The inactivation level of the HTB140 human melanoma cells after protons, FM or DTIC treatments was not enhanced by their combined application. PMID:19358719

Effects of fotemustine or dacarbasine on a melanoma cell line pretreated with therapeutic proton irradiation.

PubMed

Ristić-Fira, Aleksandra M; Korićanac, Lela B; Zakula, Jelena J; Valastro, Lucia M; Iannolo, Gioacchin; Privitera, Giuseppe; Cuttone, Giacomo; Petrović, Ivan M

2009-04-09

Considering that HTB140 melanoma cells have shown a poor response to either protons or alkylating agents, the effects of a combined use of these agents have been analysed. Cells were irradiated in the middle of the therapeutic 62 MeV proton spread out Bragg peak (SOBP). Irradiation doses were 12 or 16 Gy and are those frequently used in proton therapy. Four days after irradiation cells were treated with fotemustine (FM) or dacarbazine (DTIC). Drug concentrations were 100 and 250 microM, values close to those that produce 50% of growth inhibition. Cell viability, proliferation, survival and cell cycle distribution were assessed 7 days after irradiation that corresponds to more than six doubling times of HTB140 cells. In this way incubation periods providing the best single effects of drugs (3 days) and protons (7 days) coincided at the same time. Single proton irradiations have reduced the number of cells to approximately 50%. FM caused stronger cell inactivation due to its high toxicity, while the effectiveness of DTIC, that was important at short term, almost vanished with the incubation of 7 days. Cellular mechanisms triggered by proton irradiation differently influenced the final effects of combined treatments. Combination of protons and FM did not improve cell inactivation level achieved by single treatments. A low efficiency of the single DTIC treatment was overcome when DTIC was introduced following proton irradiation, giving better inhibitory effects with respect to the single treatments. Most of the analysed cells were in G1/S phase, viable, active and able to replicate DNA. The obtained results are the consequence of a high resistance of HTB140 melanoma cells to protons and/or drugs. The inactivation level of the HTB140 human melanoma cells after protons, FM or DTIC treatments was not enhanced by their combined application.

Single Cell Transfection through Precise Microinjection with Quantitatively Controlled Injection Volumes

NASA Astrophysics Data System (ADS)

Chow, Yu Ting; Chen, Shuxun; Wang, Ran; Liu, Chichi; Kong, Chi-Wing; Li, Ronald A.; Cheng, Shuk Han; Sun, Dong

2016-04-01

Cell transfection is a technique wherein foreign genetic molecules are delivered into cells. To elucidate distinct responses during cell genetic modification, methods to achieve transfection at the single-cell level are of great value. Herein, we developed an automated micropipette-based quantitative microinjection technology that can deliver precise amounts of materials into cells. The developed microinjection system achieved precise single-cell microinjection by pre-patterning cells in an array and controlling the amount of substance delivered based on injection pressure and time. The precision of the proposed injection technique was examined by comparing the fluorescence intensities of fluorescent dye droplets with a standard concentration and water droplets with a known injection amount of the dye in oil. Injection of synthetic modified mRNA (modRNA) encoding green fluorescence proteins or a cocktail of plasmids encoding green and red fluorescence proteins into human foreskin fibroblast cells demonstrated that the resulting green fluorescence intensity or green/red fluorescence intensity ratio were well correlated with the amount of genetic material injected into the cells. Single-cell transfection via the developed microinjection technique will be of particular use in cases where cell transfection is challenging and genetically modified of selected cells are desired.

Single Cell Transfection through Precise Microinjection with Quantitatively Controlled Injection Volumes.

PubMed

Chow, Yu Ting; Chen, Shuxun; Wang, Ran; Liu, Chichi; Kong, Chi-Wing; Li, Ronald A; Cheng, Shuk Han; Sun, Dong

2016-04-12

Cell transfection is a technique wherein foreign genetic molecules are delivered into cells. To elucidate distinct responses during cell genetic modification, methods to achieve transfection at the single-cell level are of great value. Herein, we developed an automated micropipette-based quantitative microinjection technology that can deliver precise amounts of materials into cells. The developed microinjection system achieved precise single-cell microinjection by pre-patterning cells in an array and controlling the amount of substance delivered based on injection pressure and time. The precision of the proposed injection technique was examined by comparing the fluorescence intensities of fluorescent dye droplets with a standard concentration and water droplets with a known injection amount of the dye in oil. Injection of synthetic modified mRNA (modRNA) encoding green fluorescence proteins or a cocktail of plasmids encoding green and red fluorescence proteins into human foreskin fibroblast cells demonstrated that the resulting green fluorescence intensity or green/red fluorescence intensity ratio were well correlated with the amount of genetic material injected into the cells. Single-cell transfection via the developed microinjection technique will be of particular use in cases where cell transfection is challenging and genetically modified of selected cells are desired.

Tracking single mRNA molecules in live cells

NASA Astrophysics Data System (ADS)

Moon, Hyungseok C.; Lee, Byung Hun; Lim, Kiseong; Son, Jae Seok; Song, Minho S.; Park, Hye Yoon

2016-06-01

mRNAs inside cells interact with numerous RNA-binding proteins, microRNAs, and ribosomes that together compose a highly heterogeneous population of messenger ribonucleoprotein (mRNP) particles. Perhaps one of the best ways to investigate the complex regulation of mRNA is to observe individual molecules. Single molecule imaging allows the collection of quantitative and statistical data on subpopulations and transient states that are otherwise obscured by ensemble averaging. In addition, single particle tracking reveals the sequence of events that occur in the formation and remodeling of mRNPs in real time. Here, we review the current state-of-the-art techniques in tagging, delivery, and imaging to track single mRNAs in live cells. We also discuss how these techniques are applied to extract dynamic information on the transcription, transport, localization, and translation of mRNAs. These studies demonstrate how single molecule tracking is transforming the understanding of mRNA regulation in live cells.

Plasmonic imaging of protein interactions with single bacterial cells.

PubMed

Syal, Karan; Wang, Wei; Shan, Xiaonan; Wang, Shaopeng; Chen, Hong-Yuan; Tao, Nongjian

2015-01-15

Quantifying the interactions of bacteria with external ligands is fundamental to the understanding of pathogenesis, antibiotic resistance, immune evasion, and mechanism of antimicrobial action. Due to inherent cell-to-cell heterogeneity in a microbial population, each bacterium interacts differently with its environment. This large variability is washed out in bulk assays, and there is a need of techniques that can quantify interactions of bacteria with ligands at the single bacterium level. In this work, we present a label-free and real-time plasmonic imaging technique to measure the binding kinetics of ligand interactions with single bacteria, and perform statistical analysis of the heterogeneity. Using the technique, we have studied interactions of antibodies with single Escherichia coli O157:H7 cells and demonstrated a capability of determining the binding kinetic constants of single live bacteria with ligands, and quantify heterogeneity in a microbial population. Copyright © 2014 Elsevier B.V. All rights reserved.

Highly sensitive and quantitative detection of rare pathogens through agarose droplet microfluidic emulsion PCR at the single-cell level.

PubMed

Zhu, Zhi; Zhang, Wenhua; Leng, Xuefei; Zhang, Mingxia; Guan, Zhichao; Lu, Jiangquan; Yang, Chaoyong James

2012-10-21

Genetic alternations can serve as highly specific biomarkers to distinguish fatal bacteria or cancer cells from their normal counterparts. However, these mutations normally exist in very rare amount in the presence of a large excess of non-mutated analogs. Taking the notorious pathogen E. coli O157:H7 as the target analyte, we have developed an agarose droplet-based microfluidic ePCR method for highly sensitive, specific and quantitative detection of rare pathogens in the high background of normal bacteria. Massively parallel singleplex and multiplex PCR at the single-cell level in agarose droplets have been successfully established. Moreover, we challenged the system with rare pathogen detection and realized the sensitive and quantitative analysis of a single E. coli O157:H7 cell in the high background of 100,000 excess normal K12 cells. For the first time, we demonstrated rare pathogen detection through agarose droplet microfluidic ePCR. Such a multiplex single-cell agarose droplet amplification method enables ultra-high throughput and multi-parameter genetic analysis of large population of cells at the single-cell level to uncover the stochastic variations in biological systems.

Phagocytosis: studies by optical tweezers and time-resolved microspectrofluorometry

NASA Astrophysics Data System (ADS)

Schneckenburger, Herbert; Sailer, Reinhard; Hendinger, Anita; Gschwend, Michael H.; Bauer, Manfred; Strauss, Wolfgang S. L.

1999-01-01

Cellular uptake of transparent Latex particles by J774A.1 mouse macrophages has been studied: First, single beads were kept within an optical light trap and located in close vicinity to individual cells. Uptake of the beads was visualized, and intrinsic fluorescence was detected in the spectral range of 420 - 530 nm. Second, time-gated fluorescence spectra of single cells were recorded at pre- selected times during one hour after cellular uptake. A rapid increase of autofluorescence and a subsequent decrease to the level of control cells within about 10 min. was measured within a time gate of 0 - 5 ns after the exciting laser pulses, and attributed to the 'free' coenzyme NAD(P)H. In contrast, fluorescence increase of NAD(P)H bound to proteins (measured within time gates of 5 - 10 ns or 10 - 15 ns) was less pronounced, and the subsequent decrease occurred within a longer period (about one hour).

Genotoxic potential of bee venom (Apis Mellifera) on human peripheral blood lymphocytes in vitro using single cell gel electrophoresis assay.

PubMed

Gajski, Goran; Garaj-Vrhovac, Vera

2008-09-01

Bee venom (BV) has been known to have therapeutic applications in traditional medicine to treat variety of diseases. It is also known that bee venom possesses anti-inflammatory and anticancer effects and that it can inhibit proliferation and induces apoptosis in cancer cells, but there is lack of information regarding genotoxicity of whole bee venom on normal human cells. In the present study, peripheral blood human lymphocytes from healthy donor were exposed in vitro to different concentration (5, 10, 25, 50 and 100 micro g/mL) of whole bee venom at different time periods (1, 6 and 24 hours). The single cell gel electrophoresis (SCGE) assay was used to evaluate the genotoxicity towards human cells. Results showed statistically significant increase in DNA damage caused in BV treated human lymphocytes compared to corresponding control cells for the tail length and tail moment. These results show that the extent of DNA damage, determined by the use of single cell gel electrophoresis is time and dose dependent. Based on the results it is clear that whole bee venom induces DNA damage and has genotoxic potential on human peripheral blood lymphocytes in vitro.

A single-cell pedigree analysis of alternative stochastic lymphocyte fates

PubMed Central

Hawkins, E. D.; Markham, J. F.; McGuinness, L. P.; Hodgkin, P. D.

2009-01-01

In contrast to most stimulated lymphocytes, B cells exposed to Toll-like receptor 9 ligands are nonself-adherent, allowing individual cells and families to be followed in vitro for up to 5 days. These B cells undergo phases typical of an adaptive response, dividing up to 6 times before losing the impetus for further growth and division and eventually dying by apoptosis. Using long-term microscopic imaging, accurate histories of individual lymphocyte fates were collected. Quantitative analysis of family relationships revealed that times to divide of siblings were strongly related but these correlations were progressively lost through consecutive divisions. A weaker, but significant, correlation was also found for death times among siblings. Division cessation is characterized by a loss of cell growth and the division in which this occurs is strongly inherited from the original founder cell and is related to the size this cell reaches before its first division. Thus, simple division-based dilution of factors synthesized during the first division may control the maximum division reached by stimulated cells. The stochastic distributions of times to divide, times to die, and divisions reached are also measured. Together, these results highlight the internal cellular mechanisms that control immune responses and provide a foundation for the development of new mathematical models that are correct at both single-cell and population levels. PMID:19633185

Automated single cell sorting and deposition in submicroliter drops

NASA Astrophysics Data System (ADS)

Salánki, Rita; Gerecsei, Tamás; Orgovan, Norbert; Sándor, Noémi; Péter, Beatrix; Bajtay, Zsuzsa; Erdei, Anna; Horvath, Robert; Szabó, Bálint

2014-08-01

Automated manipulation and sorting of single cells are challenging, when intact cells are needed for further investigations, e.g., RNA or DNA sequencing. We applied a computer controlled micropipette on a microscope admitting 80 PCR (Polymerase Chain Reaction) tubes to be filled with single cells in a cycle. Due to the Laplace pressure, fluid starts to flow out from the micropipette only above a critical pressure preventing the precise control of drop volume in the submicroliter range. We found an anomalous pressure additive to the Laplace pressure that we attribute to the evaporation of the drop. We have overcome the problem of the critical dropping pressure with sequentially operated fast fluidic valves timed with a millisecond precision. Minimum drop volume was 0.4-0.7 μl with a sorting speed of 15-20 s per cell. After picking NE-4C neuroectodermal mouse stem cells and human primary monocytes from a standard plastic Petri dish we could gently deposit single cells inside tiny drops. 94 ± 3% and 54 ± 7% of the deposited drops contained single cells for NE-4C and monocytes, respectively. 7.5 ± 4% of the drops contained multiple cells in case of monocytes. Remaining drops were empty. Number of cells deposited in a drop could be documented by imaging the Petri dish before and after sorting. We tuned the adhesion force of cells to make the manipulation successful without the application of microstructures for trapping cells on the surface. We propose that our straightforward and flexible setup opens an avenue for single cell isolation, critically needed for the rapidly growing field of single cell biology.

Single-cell RNA-sequencing: The future of genome biology is now

PubMed Central

Picelli, Simone

2017-01-01

ABSTRACT Genome-wide single-cell analysis represents the ultimate frontier of genomics research. In particular, single-cell RNA-sequencing (scRNA-seq) studies have been boosted in the last few years by an explosion of new technologies enabling the study of the transcriptomic landscape of thousands of single cells in complex multicellular organisms. More sensitive and automated methods are being continuously developed and promise to deliver better data quality and higher throughput with less hands-on time. The outstanding amount of knowledge that is going to be gained from present and future studies will have a profound impact in many aspects of our society, from the introduction of truly tailored cancer treatments, to a better understanding of antibiotic resistance and host-pathogen interactions; from the discovery of the mechanisms regulating stem cell differentiation to the characterization of the early event of human embryogenesis. PMID:27442339

Emerging Imaging and Genomic Tools for Developmental Systems Biology.

PubMed

Liu, Zhe; Keller, Philipp J

2016-03-21

Animal development is a complex and dynamic process orchestrated by exquisitely timed cell lineage commitment, divisions, migration, and morphological changes at the single-cell level. In the past decade, extensive genetic, stem cell, and genomic studies provided crucial insights into molecular underpinnings and the functional importance of genetic pathways governing various cellular differentiation processes. However, it is still largely unknown how the precise coordination of these pathways is achieved at the whole-organism level and how the highly regulated spatiotemporal choreography of development is established in turn. Here, we discuss the latest technological advances in imaging and single-cell genomics that hold great promise for advancing our understanding of this intricate process. We propose an integrated approach that combines such methods to quantitatively decipher in vivo cellular dynamic behaviors and their underlying molecular mechanisms at the systems level with single-cell, single-molecule resolution. Copyright © 2016 Elsevier Inc. All rights reserved.

Flow analysis of human chromosome sets by means of mixing-stirring device

NASA Astrophysics Data System (ADS)

Zenin, Valeri V.; Aksenov, Nicolay D.; Shatrova, Alla N.; Klopov, Nicolay V.; Cram, L. Scott; Poletaev, Andrey I.

1997-05-01

A new mixing and stirring device (MSD) was used to perform flow karyotype analysis of single human mitotic chromosomes analyzed so as to maintain the identity of chromosomes derived from the same cell. An improved method for cell preparation and intracellular staining of chromosomes was developed. The method includes enzyme treatment, incubation with saponin and separation of prestained cells from debris on a sucrose gradient. Mitotic cells are injected one by one in the MSD which is located inside the flow chamber where cells are ruptured, thereby releasing chromosomes. The set of chromosomes proceeds to flow in single file fashion to the point of analysis. The device works in a stepwise manner. The concentration of cells in the sample must be kept low to ensure that only one cell at a time enters the breaking chamber. Time-gated accumulation of data in listmode files makes it possible to separate chromosome sets comprising of single cells. The software that was developed classifies chromosome sets according to different criteria: total number of chromosomes, overall DNA content in the set, and the number of chromosomes of certain types. This approach combines the high performance of flow cytometry with the advantages of image analysis. Examples obtained with different human cell lines are presented.

Dissecting genomic diversity, one cell at a time

PubMed Central

Blainey, Paul C; Quake, Stephen R

2014-01-01

Emerging technologies are bringing single-cell genome sequencing into the mainstream; this field has already yielded insights into the genetic architecture and variability between cells that highlight the dynamic nature of the genome. PMID:24524132

Metabolic oxygen consumption measurement with a single-cell biosensor after particle microbeam irradiation

PubMed Central

Zhang, Bo; Messerli, Mark; Randers-Pehrson, Gerhard; Hei, Tom K.; Brenner, David J.

2015-01-01

A noninvasive, self-referencing biosensor/probe system has been integrated into the Columbia University Radiological Research Accelerator Facility Microbeam II end station. A single-cell oxygen consumption measurement has been conducted with this type of oxygen probe in 37°C Krebs–Ringer Bicarbonate buffer immediately before and after a single-cell microbeam irradiation. It is the first such measurement made for a microbeam irradiation, and a six fold increment of oxygen flux induced during a 15-s period of time has been observed following radiation exposure. The experimental procedure and the results are discussed. PMID:25335641

Single Cell Spectroscopy: Noninvasive Measures of Small-Scale Structure and Function

PubMed Central

Mousoulis, Charilaos; Xu, Xin; Reiter, David A.; Neu, Corey P.

2013-01-01

The advancement of spectroscopy methods attained through increases in sensitivity, and often with the coupling of complementary techniques, has enabled real-time structure and function measurements of single cells. The purpose of this review is to illustrate, in light of advances, the strengths and the weaknesses of these methods. Included also is an assessment of the impact of the experimental setup and conditions of each method on cellular function and integrity. A particular emphasis is placed on noninvasive and nondestructive techniques for achieving single cell detection, including nuclear magnetic resonance, in addition to physical, optical, and vibrational methods. PMID:23886910

Cell fixation and preservation for droplet-based single-cell transcriptomics.

PubMed

Alles, Jonathan; Karaiskos, Nikos; Praktiknjo, Samantha D; Grosswendt, Stefanie; Wahle, Philipp; Ruffault, Pierre-Louis; Ayoub, Salah; Schreyer, Luisa; Boltengagen, Anastasiya; Birchmeier, Carmen; Zinzen, Robert; Kocks, Christine; Rajewsky, Nikolaus

2017-05-19

Recent developments in droplet-based microfluidics allow the transcriptional profiling of thousands of individual cells in a quantitative, highly parallel and cost-effective way. A critical, often limiting step is the preparation of cells in an unperturbed state, not altered by stress or ageing. Other challenges are rare cells that need to be collected over several days or samples prepared at different times or locations. Here, we used chemical fixation to address these problems. Methanol fixation allowed us to stabilise and preserve dissociated cells for weeks without compromising single-cell RNA sequencing data. By using mixtures of fixed, cultured human and mouse cells, we first showed that individual transcriptomes could be confidently assigned to one of the two species. Single-cell gene expression from live and fixed samples correlated well with bulk mRNA-seq data. We then applied methanol fixation to transcriptionally profile primary cells from dissociated, complex tissues. Low RNA content cells from Drosophila embryos, as well as mouse hindbrain and cerebellum cells prepared by fluorescence-activated cell sorting, were successfully analysed after fixation, storage and single-cell droplet RNA-seq. We were able to identify diverse cell populations, including neuronal subtypes. As an additional resource, we provide 'dropbead', an R package for exploratory data analysis, visualization and filtering of Drop-seq data. We expect that the availability of a simple cell fixation method will open up many new opportunities in diverse biological contexts to analyse transcriptional dynamics at single-cell resolution.

Discrepancies between patterns of potentially lethal damage repair in the RIF-1 tumor system in vitro and in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rasey, J.S.; Nelson, N.J.

1983-01-01

Repair of potentially lethal damage (PLD) was studied in the RIF-1 tumor system in several different growth states in vivo. Exponentially growing, fed plateau, and unfed plateau cells in cell culture as well as small and large subcutaneous or intramuscular tumors were investigated. Large single doses of radiation followed by variable repair times as well as graded doses of radiation to generate survival curves immediately after irradiation or after full repair were investigated. All repair-promoting conditions studied in vitro (delayed subculture, exposure of cells to depleted growth medium after irradiation) increased surviving fraction after a single dose. The D/sub 0/more » of the cell survival curve was also increased by these procedures. No PLD repair was observed for any tumors irradiated in vivo and maintained in the animal for varying times prior to assay in vitro. The nearly 100% cell yield obtained when this tumor is prepared as a single-cell suspension for colony formation, the representative cell sample obtained, and the constant cell yield per gram as a function of time postirradiation suggest that this discrepancy is not an artifact of the assay system. The most logical explanation of these data and information on radiocurability of this neoplasm is that PLD repair, which is so frequently demonstrated in vitro, may not be a major factor in the radioresponse of this tumor when left in situ.« less

Discrepancies between patterns of potentially lethal damage repair in the RIF-1 tumor system in vitro and in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rasey, J.S.; Nelson, N.J.

1983-01-01

Repair of potentially lethal damage (PLD) was studied in the RIF-1 tumor system in several different growth states in vivo and in vitro. Exponentially growing, fed plateau, and unfed plateau cells in cell culture as well as small and large subcutaneous or intramuscular tumors were investigated. Large single doses of radiation followed by variable repair times as well as graded doses of radiation to generate survival curves immediately after irradiation or after full repair were investigated. All repair-promoting conditions studied in vitro (delayed subculture, exposure of cells to depleted growth medium after irradiation) increased surviving fraction after a single dose.more » The D0 of the cell survival curve was also increased by these procedures. No PLD repair was observed for any tumors irradiated in vivo and maintained in the animal for varying times prior to assay in vitro. The nearly 100% cell yield obtained when this tumor is prepared as a single-cell suspension for colony formation, the representative cell sample obtained, and the constant cell yield per gram as a function of time postirradiation suggest that this discrepancy is not an artifact of the assay system. The most logical explanation of these data and information on radiocurability of this neoplasm is that PLD repair, which is so frequently demonstrated in vitro, may not be a major factor in the radioresponse of this tumor when left in situ.« less

Changes in the biomechanical properties of a single cell induced by nonthermal atmospheric pressure micro-dielectric barrier discharge plasma.

PubMed

Choi, Hyeongwon; Choi, Eun Ha; Kim, Kyung Sook

2017-10-01

Mechanical properties of a single cell are closely related to the fate and functions of the cell. Changes in mechanical properties may cause diseases or cell apoptosis. Selective cytotoxic effects of nonthermal atmospheric pressure micro-dielectric barrier discharge (DBD) plasma have been demonstrated on cancer cells. In this work, changes in the mechanical properties of a single cell induced by nonthermal atmospheric pressure micro-DBD plasma were investigated using atomic force microscopy (AFM). Two cervical cancer cell lines (HeLa and SiHa) and normal human fibroblast cells (HFBs) were exposed to micro-DBD plasma for various exposure times. The elasticity of a single cell was determined by force-distance curve measurement using AFM. Young's modulus was decreased by plasma treatment for all cells. The Young's modulus of plasma-treated HeLa cells was decreased by 75% compared to nontreated HeLa cells. In SiHa cells and HFBs, elasticity was decreased slightly. Chemical changes induced by the plasma treatment, which were observed by Raman spectroscopy, were also significant in HeLa cells compared to SiHa cells and HFBs. These results suggested that the molecular changes induced by micro-DBD plasma were related to cell mechanical changes. © 2017 Wiley Periodicals, Inc.

An automated approach for single-cell tracking in epifluorescence microscopy applied to E. coli growth analysis on microfluidics biochips

NASA Astrophysics Data System (ADS)

Fetita, Catalin; Kirov, Boris; Jaramillo, Alfonso; Lefevre, Christophe

2012-03-01

With the accumulation of knowledge for the intimate molecular mechanisms governing the processes inside the living cells in the later years, the ability to characterize the performance of elementary genetic circuits and parts at the single-cell level is becoming of crucial importance. Biological science is arriving to the point where it can develop hypothesis for the action of each molecule participating in the biochemical reactions and need proper techniques to test those hypothesis. Microfluidics is emerging as the technology that combined with high-magnification microscopy will allow for the long-term single-cell level observation of bacterial physiology. In this study we design, build and characterize the gene dynamics of genetic circuits as one of the basic parts governing programmed cell behavior. We use E. coli as model organism and grow it in microfluidics chips, which we observe with epifluorescence microscopy. One of the most invaluable segments of this technology is the consequent image processing, since it allows for the automated analysis of vast amount of single-cell observation and the fast and easy derivation of conclusions based on that data. Specifically, we are interested in promoter activity as function of time. We expect it to be oscillatory and for that we use GFP (green fluorescent protein) as a reporter in our genetic circuits. In this paper, an automated framework for single-cell tracking in phase-contrast microscopy is developed, combining 2D segmentation of cell time frames and graph-based reconstruction of their spatiotemporal evolution with fast tracking of the associated fluorescence signal. The results obtained on the investigated biological database are presented and discussed.

Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

DOE Office of Scientific and Technical Information (OSTI.GOV)

Horita, Nobukatsu; Tsuchiya, Kiichiro, E-mail: kii.gast@tmd.ac.jp; Hayashi, Ryohei

Highlights: • Lentivirus mixed with Matrigel enables direct infection of intestinal organoids. • Our original approach allows the marking of a single stem cell in a crypt. • Time-lapse imaging shows the dynamics of a single stem cell. • Our lentivirus transgene system demonstrates plural long-lived stem cells in a crypt. - Abstract: Background and aims: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualisedmore » in the same colour. Methods: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. Results: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. Conclusions: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.« less

Fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for accurate detection and counting of RNA copies in single cells

DOE PAGES

Cui, Yi; Hu, Dehong; Markillie, Lye Meng; ...

2017-10-04

Here, quantitative gene expression analysis in intact single cells can be achieved using single molecule-based fluorescence in situ hybridization (smFISH). This approach relies on fluorescence intensity to distinguish between true signals, emitted from an RNA copy hybridized with multiple oligonucleotide probes, and background noise. Thus, the precision in smFISH is often compromised by partial or nonspecific probe binding and tissue autofluorescence, especially when only a small number of probes can be fitted to the target transcript. Here we provide an accurate approach for setting quantitative thresholds between true and false signals, which relies on on-off duty cycles of photoswitchable dyes.more » This fluctuation localization imaging-based FISH (fliFISH) uses on-time fractions (measured over a series of exposures) collected from transcripts bound to as low as 8 probes, which are distinct from on-time fractions collected from nonspecifically bound probes or autofluorescence. Using multicolor fliFISH, we identified radial gene expression patterns in mouse pancreatic islets for insulin, the transcription factor, NKX2-2 and their ratio ( Nkx2- 2/Ins2). These radial patterns, showing higher values in β cells at the islet core and lower values in peripheral cells, were lost in diabetic mouse islets. In summary, fliFISH provides an accurate, quantitative approach for detecting and counting true RNA copies and rejecting false signals by their distinct on-time fractions, laying the foundation for reliable single-cell transcriptomics.« less

Fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for accurate detection and counting of RNA copies in single cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cui, Yi; Hu, Dehong; Markillie, Lye Meng

Here, quantitative gene expression analysis in intact single cells can be achieved using single molecule-based fluorescence in situ hybridization (smFISH). This approach relies on fluorescence intensity to distinguish between true signals, emitted from an RNA copy hybridized with multiple oligonucleotide probes, and background noise. Thus, the precision in smFISH is often compromised by partial or nonspecific probe binding and tissue autofluorescence, especially when only a small number of probes can be fitted to the target transcript. Here we provide an accurate approach for setting quantitative thresholds between true and false signals, which relies on on-off duty cycles of photoswitchable dyes.more » This fluctuation localization imaging-based FISH (fliFISH) uses on-time fractions (measured over a series of exposures) collected from transcripts bound to as low as 8 probes, which are distinct from on-time fractions collected from nonspecifically bound probes or autofluorescence. Using multicolor fliFISH, we identified radial gene expression patterns in mouse pancreatic islets for insulin, the transcription factor, NKX2-2 and their ratio ( Nkx2- 2/Ins2). These radial patterns, showing higher values in β cells at the islet core and lower values in peripheral cells, were lost in diabetic mouse islets. In summary, fliFISH provides an accurate, quantitative approach for detecting and counting true RNA copies and rejecting false signals by their distinct on-time fractions, laying the foundation for reliable single-cell transcriptomics.« less

Kinetics of Cell Fusion Induced by a Syncytia-Producing Mutant of Herpes Simplex Virus Type I

PubMed Central

Person, Stanley; Knowles, Robert W.; Read, G. Sullivan; Warner, Susan C.; Bond, Vincent C.

1976-01-01

We have isolated a number of plaque-morphology mutants from a strain of herpes simplex virus type I which, unlike the wild type, cause extensive cell fusion during a productive viral infection. After the onset of fusion, there is an exponential decrease in the number of single cells as a function of time after infection. At a multiplicity of infection (MOI) of 3.8 plaque-forming units per cell, fusion begins 5.3 h after infection with the number of single cells decreasing to 10% of the original number 10.2 h after infection. As the MOI is gradually increased from 0.4 to 8, the onset of fusion occurs earlier during infection. However, when the MOI is increased from 8 to 86, the onset of fusion does not occur any earlier. The rate of fusion is independent of the MOI for an MOI greater than 1. The rate of fusion varies linearly with initial cell density up to 3.5 × 104 cells/cm2 and is independent of initial cell density at higher cell concentrations. To assay cell fusion we have developed a simple quantitative assay using a Coulter counter to measure the number of single cells as a function of time after infection. Data obtained using a Coulter counter are similar to those obtained with a microscope assay. PMID:173881

Single-molecule analysis of steroid receptor and cofactor action in living cells

PubMed Central

Paakinaho, Ville; Presman, Diego M.; Ball, David A.; Johnson, Thomas A.; Schiltz, R. Louis; Levitt, Peter; Mazza, Davide; Morisaki, Tatsuya; Karpova, Tatiana S.; Hager, Gordon L.

2017-01-01

Population-based assays have been employed extensively to investigate the interactions of transcription factors (TFs) with chromatin and are often interpreted in terms of static and sequential binding. However, fluorescence microscopy techniques reveal a more dynamic binding behaviour of TFs in live cells. Here we analyse the strengths and limitations of in vivo single-molecule tracking and performed a comprehensive analysis on the intranuclear dwell times of four steroid receptors and a number of known cofactors. While the absolute residence times estimates can depend on imaging acquisition parameters due to sampling bias, our results indicate that only a small proportion of factors are specifically bound to chromatin at any given time. Interestingly, the glucocorticoid receptor and its cofactors affect each other’s dwell times in an asymmetric manner. Overall, our data indicate transient rather than stable TF-cofactors chromatin interactions at response elements at the single-molecule level. PMID:28635963

Microfluidics-based, time-resolved mechanical phenotyping of cells using high-speed imaging

NASA Astrophysics Data System (ADS)

Belotti, Yuri; Conneely, Michael; Huang, Tianjun; McKenna, Stephen; Nabi, Ghulam; McGloin, David

2017-07-01

We demonstrate a single channel hydrodynamic stretching microfluidic device that relies on high-speed imaging to allow repeated dynamic cell deformation measurements. Experiments on prostate cancer cells suggest richer data than current approaches.

Ring-Shaped Microlanes and Chemical Barriers as a Platform for Probing Single-Cell Migration.

PubMed

Schreiber, Christoph; Segerer, Felix J; Wagner, Ernst; Roidl, Andreas; Rädler, Joachim O

2016-05-31

Quantification and discrimination of pharmaceutical and disease-related effects on cell migration requires detailed characterization of single-cell motility. In this context, micropatterned substrates that constrain cells within defined geometries facilitate quantitative readout of locomotion. Here, we study quasi-one-dimensional cell migration in ring-shaped microlanes. We observe bimodal behavior in form of alternating states of directional migration (run state) and reorientation (rest state). Both states show exponential lifetime distributions with characteristic persistence times, which, together with the cell velocity in the run state, provide a set of parameters that succinctly describe cell motion. By introducing PEGylated barriers of different widths into the lane, we extend this description by quantifying the effects of abrupt changes in substrate chemistry on migrating cells. The transit probability decreases exponentially as a function of barrier width, thus specifying a characteristic penetration depth of the leading lamellipodia. Applying this fingerprint-like characterization of cell motion, we compare different cell lines, and demonstrate that the cancer drug candidate salinomycin affects transit probability and resting time, but not run time or run velocity. Hence, the presented assay allows to assess multiple migration-related parameters, permits detailed characterization of cell motility, and has potential applications in cell biology and advanced drug screening.

Single-cell imaging of the heat-shock response in colon cancer cells suggests that magnitude and length rather than time of onset determines resistance to apoptosis.

PubMed

Ramapathiran, Lavanya; Bernas, Tytus; Walter, Franziska; Williams, Linda; Düssmann, Heiko; Concannon, Caoimhín G; Prehn, Jochen H M

2014-02-01

Targeting the proteasome is a valuable approach for cancer therapy, potentially limited by pro-survival pathways that are induced in parallel to cell death. Whether these pro-survival pathways are activated in all cells, show different activation kinetics in sensitive versus resistant cells or interact functionally with cell death pathways is unknown. We monitored activation of the heat-shock response (HSR), a key survival pathway induced by proteasome inhibition, relative to apoptosis activation in HCT116 colon cancer cells expressing enhanced green fluorescent protein (EGFP) under the control of the HSP70 promoter. Single-cell and high-content time-lapse imaging of epoxomicin treatment revealed that neither basal activity nor the time of onset of the HSR differed between resistant and sensitive populations. However, resistant cells had significantly higher and prolonged reporter activity than those that succumbed to cell death. p53 deficiency protected against cell death but failed to modulate the HSR. By contrast, inhibition of the HSR significantly increased the cytotoxicity of epoxomicin. Our data provide novel insights into the kinetics and heterogeneity of the HSR during proteasome inhibition, suggesting that the HSR modulates cell death signalling unidirectionally.

Cell-Specific Multifunctional Processing of Heterogeneous Cell Systems in a Single Laser Pulse Treatment

PubMed Central

Lukianova-Hleb, Ekaterina Y.; Mutonga, Martin B. G.; Lapotko, Dmitri O.

2012-01-01

Current methods of cell processing for gene and cell therapies use several separate procedures for gene transfer and cell separation or elimination, because no current technology can offer simultaneous multi-functional processing of specific cell sub-sets in highly heterogeneous cell systems. Using the cell-specific generation of plasmonic nanobubbles of different sizes around cell-targeted gold nanoshells and nanospheres, we achieved simultaneous multifunctional cell-specific processing in a rapid single 70 ps laser pulse bulk treatment of heterogeneous cell suspension. This method supported the detection of cells, delivery of external molecular cargo to one type of cells and the concomitant destruction of another type of cells without damaging other cells in suspension, and real-time guidance of the two above cellular effects. PMID:23167546

Prospective guidance in a free-swimming cell.

PubMed

Delafield-Butt, Jonathan T; Pepping, Gert-Jan; McCaig, Colin D; Lee, David N

2012-07-01

A systems theory of movement control in animals is presented in this article and applied to explaining the controlled behaviour of the single-celled Paramecium caudatum in an electric field. The theory-General Tau Theory-is founded on three basic principles: (i) all purposive movement entails prospectively controlling the closure of action-gaps (e.g. a distance gap when reaching, or an angle gap when steering); (ii) the sole informational variable required for controlling gaps is the relative rate of change of the gap (the time derivative of the gap size divided by the size), which can be directly sensed; and (iii) a coordinated movement is achieved by keeping the relative rates of change of gaps in a constant ratio. The theory is supported by studies of controlled movement in mammals, birds and insects. We now show for the first time that it is also supported by single-celled paramecia steering to the cathode in a bi-polar electric field. General Tau Theory is deployed to explain this guided steering by the cell. This article presents the first computational model of prospective perceptual control in a non-neural, single-celled system.

Automated profiling of individual cell-cell interactions from high-throughput time-lapse imaging microscopy in nanowell grids (TIMING).

PubMed

Merouane, Amine; Rey-Villamizar, Nicolas; Lu, Yanbin; Liadi, Ivan; Romain, Gabrielle; Lu, Jennifer; Singh, Harjeet; Cooper, Laurence J N; Varadarajan, Navin; Roysam, Badrinath

2015-10-01

There is a need for effective automated methods for profiling dynamic cell-cell interactions with single-cell resolution from high-throughput time-lapse imaging data, especially, the interactions between immune effector cells and tumor cells in adoptive immunotherapy. Fluorescently labeled human T cells, natural killer cells (NK), and various target cells (NALM6, K562, EL4) were co-incubated on polydimethylsiloxane arrays of sub-nanoliter wells (nanowells), and imaged using multi-channel time-lapse microscopy. The proposed cell segmentation and tracking algorithms account for cell variability and exploit the nanowell confinement property to increase the yield of correctly analyzed nanowells from 45% (existing algorithms) to 98% for wells containing one effector and a single target, enabling automated quantification of cell locations, morphologies, movements, interactions, and deaths without the need for manual proofreading. Automated analysis of recordings from 12 different experiments demonstrated automated nanowell delineation accuracy >99%, automated cell segmentation accuracy >95%, and automated cell tracking accuracy of 90%, with default parameters, despite variations in illumination, staining, imaging noise, cell morphology, and cell clustering. An example analysis revealed that NK cells efficiently discriminate between live and dead targets by altering the duration of conjugation. The data also demonstrated that cytotoxic cells display higher motility than non-killers, both before and during contact. broysam@central.uh.edu or nvaradar@central.uh.edu Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Spatially coordinated dynamic gene transcription in living pituitary tissue

PubMed Central

Featherstone, Karen; Hey, Kirsty; Momiji, Hiroshi; McNamara, Anne V; Patist, Amanda L; Woodburn, Joanna; Spiller, David G; Christian, Helen C; McNeilly, Alan S; Mullins, John J; Finkenstädt, Bärbel F; Rand, David A; White, Michael RH; Davis, Julian RE

2016-01-01

Transcription at individual genes in single cells is often pulsatile and stochastic. A key question emerges regarding how this behaviour contributes to tissue phenotype, but it has been a challenge to quantitatively analyse this in living cells over time, as opposed to studying snap-shots of gene expression state. We have used imaging of reporter gene expression to track transcription in living pituitary tissue. We integrated live-cell imaging data with statistical modelling for quantitative real-time estimation of the timing of switching between transcriptional states across a whole tissue. Multiple levels of transcription rate were identified, indicating that gene expression is not a simple binary ‘on-off’ process. Immature tissue displayed shorter durations of high-expressing states than the adult. In adult pituitary tissue, direct cell contacts involving gap junctions allowed local spatial coordination of prolactin gene expression. Our findings identify how heterogeneous transcriptional dynamics of single cells may contribute to overall tissue behaviour. DOI: http://dx.doi.org/10.7554/eLife.08494.001 PMID:26828110

Single-cell analysis of HIV-1 transcriptional activity reveals expression of proviruses in expanded clones during ART.

PubMed

Wiegand, Ann; Spindler, Jonathan; Hong, Feiyu F; Shao, Wei; Cyktor, Joshua C; Cillo, Anthony R; Halvas, Elias K; Coffin, John M; Mellors, John W; Kearney, Mary F

2017-05-02

Little is known about the fraction of human immunodeficiency virus type 1 (HIV-1) proviruses that express unspliced viral RNA in vivo or about the levels of HIV RNA expression within single infected cells. We developed a sensitive cell-associated HIV RNA and DNA single-genome sequencing (CARD-SGS) method to investigate fractional proviral expression of HIV RNA (1.3-kb fragment of p6, protease, and reverse transcriptase) and the levels of HIV RNA in single HIV-infected cells from blood samples obtained from individuals with viremia or individuals on long-term suppressive antiretroviral therapy (ART). Spiking experiments show that the CARD-SGS method can detect a single cell expressing HIV RNA. Applying CARD-SGS to blood mononuclear cells in six samples from four HIV-infected donors (one with viremia and not on ART and three with viremia suppressed on ART) revealed that an average of 7% of proviruses (range: 2-18%) expressed HIV RNA. Levels of expression varied from one to 62 HIV RNA molecules per cell (median of 1). CARD-SGS also revealed the frequent expression of identical HIV RNA sequences across multiple single cells and across multiple time points in donors on suppressive ART consistent with constitutive expression of HIV RNA in infected cell clones. Defective proviruses were found to express HIV RNA at levels similar to those proviruses that had no obvious defects. CARD-SGS is a useful tool to characterize fractional proviral expression in single infected cells that persist despite ART and to assess the impact of experimental interventions on proviral populations and their expression.

Effects of methotrexate on the viscoelastic properties of single cells probed by atomic force microscopy.

PubMed

Li, Mi; Liu, Lianqing; Xiao, Xiubin; Xi, Ning; Wang, Yuechao

2016-10-01

Methotrexate is a commonly used anti-cancer chemotherapy drug. Cellular mechanical properties are fundamental parameters that reflect the physiological state of a cell. However, so far the role of cellular mechanical properties in the actions of methotrexate is still unclear. In recent years, probing the behaviors of single cells with the use of atomic force microscopy (AFM) has contributed much to the field of cell biomechanics. In this work, with the use of AFM, the effects of methotrexate on the viscoelastic properties of four types of cells were quantitatively investigated. The inhibitory and cytotoxic effects of methotrexate on the proliferation of cells were observed by optical and fluorescence microscopy. AFM indenting was used to measure the changes of cellular viscoelastic properties (Young's modulus and relaxation time) by using both conical tip and spherical tip, quantitatively showing that the stimulation of methotrexate resulted in a significant decrease of both cellular Young's modulus and relaxation times. The morphological changes of cells induced by methotrexate were visualized by AFM imaging. The study improves our understanding of methotrexate action and offers a novel way to quantify drug actions at the single-cell level by measuring cellular viscoelastic properties, which may have potential impacts on developing label-free methods for drug evaluation.

Cell cycle tracking for irradiated and unirradiated bystander cells in a single colony with exposure to a soft X-ray microbeam.

PubMed

Kaminaga, Kiichi; Noguchi, Miho; Narita, Ayumi; Hattori, Yuya; Usami, Noriko; Yokoya, Akinari

2016-11-01

To establish a new experimental technique to explore the photoelectric and subsequent Auger effects on the cell cycles of soft X-ray microbeam-irradiated cells and unirradiated bystander cells in a single colony. Several cells located in the center of a microcolony of HeLa-Fucci cells consisting of 20-80 cells were irradiated with soft X-ray (5.35 keV) microbeam using synchrotron radiation as a light source. All cells in the colony were tracked for 72 h by time-lapse microscopy imaging. Cell cycle progression, division, and death of each cell in the movies obtained were analyzed by pedigree assay. The number of cell divisions in the microcolony was also determined. The fates of these cells were clarified by tracking both irradiated and unirradiated bystander cells. Irradiated cells showed significant cell cycle retardation, explosive cell death, or cell fusion after a few divisions. These serious effects were also observed in 15 and 26% of the bystander cells for 10 and 20 Gy irradiation, respectively, and frequently appeared in at least two daughter or granddaughter cells from a single-parent cell. We successfully tracked the fates of microbeam-irradiated cells and unirradiated bystander cells with live cell recordings, which have revealed the dynamics of soft X-ray irradiated and unirradiated bystander cells for the first time. Notably, cell deaths or cell cycle arrests frequently arose in closely related cells. These details would not have been revealed by a conventional immunostaining imaging method. Our approach promises to reveal the dynamic cellular effects of soft X-ray microbeam irradiation and subsequent Auger processes from various endpoints in future studies.

Chemotaxis of Cell Populations through Confined Spaces at Single-Cell Resolution

PubMed Central

Tong, ZiQiu; Balzer, Eric M.; Dallas, Matthew R.; Hung, Wei-Chien; Stebe, Kathleen J.; Konstantopoulos, Konstantinos

2012-01-01

Cell migration is crucial for both physiological and pathological processes. Current in vitro cell motility assays suffer from various drawbacks, including insufficient temporal and/or optical resolution, or the failure to include a controlled chemotactic stimulus. Here, we address these limitations with a migration chamber that utilizes a self-sustaining chemotactic gradient to induce locomotion through confined environments that emulate physiological settings. Dynamic real-time analysis of both population-scale and single-cell movement are achieved at high resolution. Interior surfaces can be functionalized through adsorption of extracellular matrix components, and pharmacological agents can be administered to cells directly, or indirectly through the chemotactic reservoir. Direct comparison of multiple cell types can be achieved in a single enclosed system to compare inherent migratory potentials. Our novel microfluidic design is therefore a powerful tool for the study of cellular chemotaxis, and is suitable for a wide range of biological and biomedical applications. PMID:22279529

Sequential Super-Resolution Imaging of Bacterial Regulatory Proteins: The Nucleoid and the Cell Membrane in Single, Fixed E. coli Cells.

PubMed

Spahn, Christoph; Glaesmann, Mathilda; Gao, Yunfeng; Foo, Yong Hwee; Lampe, Marko; Kenney, Linda J; Heilemann, Mike

2017-01-01

Despite their small size and the lack of compartmentalization, bacteria exhibit a striking degree of cellular organization, both in time and space. During the last decade, a group of new microscopy techniques emerged, termed super-resolution microscopy or nanoscopy, which facilitate visualizing the organization of proteins in bacteria at the nanoscale. Single-molecule localization microscopy (SMLM) is especially well suited to reveal a wide range of new information regarding protein organization, interaction, and dynamics in single bacterial cells. Recent developments in click chemistry facilitate the visualization of bacterial chromatin with a resolution of ~20 nm, providing valuable information about the ultrastructure of bacterial nucleoids, especially at short generation times. In this chapter, we describe a simple-to-realize protocol that allows determining precise structural information of bacterial nucleoids in fixed cells, using direct stochastic optical reconstruction microscopy (dSTORM). In combination with quantitative photoactivated localization microscopy (PALM), the spatial relationship of proteins with the bacterial chromosome can be studied. The position of a protein of interest with respect to the nucleoids and the cell cylinder can be visualized by super-resolving the membrane using point accumulation for imaging in nanoscale topography (PAINT). The combination of the different SMLM techniques in a sequential workflow maximizes the information that can be extracted from single cells, while maintaining optimal imaging conditions for each technique.

Tracing the origin of disseminated tumor cells in breast cancer using single-cell sequencing.

PubMed

Demeulemeester, Jonas; Kumar, Parveen; Møller, Elen K; Nord, Silje; Wedge, David C; Peterson, April; Mathiesen, Randi R; Fjelldal, Renathe; Zamani Esteki, Masoud; Theunis, Koen; Fernandez Gallardo, Elia; Grundstad, A Jason; Borgen, Elin; Baumbusch, Lars O; Børresen-Dale, Anne-Lise; White, Kevin P; Kristensen, Vessela N; Van Loo, Peter; Voet, Thierry; Naume, Bjørn

2016-12-09

Single-cell micro-metastases of solid tumors often occur in the bone marrow. These disseminated tumor cells (DTCs) may resist therapy and lay dormant or progress to cause overt bone and visceral metastases. The molecular nature of DTCs remains elusive, as well as when and from where in the tumor they originate. Here, we apply single-cell sequencing to identify and trace the origin of DTCs in breast cancer. We sequence the genomes of 63 single cells isolated from six non-metastatic breast cancer patients. By comparing the cells' DNA copy number aberration (CNA) landscapes with those of the primary tumors and lymph node metastasis, we establish that 53% of the single cells morphologically classified as tumor cells are DTCs disseminating from the observed tumor. The remaining cells represent either non-aberrant "normal" cells or "aberrant cells of unknown origin" that have CNA landscapes discordant from the tumor. Further analyses suggest that the prevalence of aberrant cells of unknown origin is age-dependent and that at least a subset is hematopoietic in origin. Evolutionary reconstruction analysis of bulk tumor and DTC genomes enables ordering of CNA events in molecular pseudo-time and traced the origin of the DTCs to either the main tumor clone, primary tumor subclones, or subclones in an axillary lymph node metastasis. Single-cell sequencing of bone marrow epithelial-like cells, in parallel with intra-tumor genetic heterogeneity profiling from bulk DNA, is a powerful approach to identify and study DTCs, yielding insight into metastatic processes. A heterogeneous population of CNA-positive cells is present in the bone marrow of non-metastatic breast cancer patients, only part of which are derived from the observed tumor lineages.

Selective Amperometric Recording of Endogenous Ascorbate Secretion from a Single Rat Adrenal Chromaffin Cell with Pretreated Carbon Fiber Microelectrodes.

PubMed

Wang, Kai; Xiao, Tongfang; Yue, Qingwei; Wu, Fei; Yu, Ping; Mao, Lanqun

2017-09-05

Quantitative description of ascorbate secretion at a single-cell level is of great importance in physiological studies; however, most studies on the ascorbate secretion have so far been performed through analyzing cell extracts with high performance liquid chromatography, which lacks time resolution and analytical performance on a single-cell level. This study demonstrates a single-cell amperometry with carbon fiber microelectrodes (CFEs) to selectively monitor amperometric vesicular secretion of endogenous ascorbate from a single rat adrenal chromaffin cell. The CFEs are electrochemically pretreated in a weakly basic solution (pH 9.5), and such pretreatment essentially enables the oxidation of ascorbate to occur at a relatively low potential (i.e., 0.0 V vs Ag/AgCl), and further a high selectivity for ascorbate measurement over endogenously existing electroactive species such as epinephrine, norepinephrine, and dopamine. The selectivity is ensured by much larger amperometric response at the pretreated CFEs toward ascorbate over those toward other endogenously existing electroactive species added into the solution or ejected to the electrode with a micropuffer pipet, and by the totally suppressed current response by adding ascorbate oxidase into the cell lysate. With the pretreated CFE-based single-cell amperometry developed here, exocytosis of endogenous ascorbate of rat adrenal chromaffin cells is directly observed and ensured with the calcium ion-dependent high K + -induced secretion of endogenous ascorbate from the cells. Moreover, the quantitative information on the exocytosis of endogenous ascorbate is provided.

Effect of contact time and force on monocyte adhesion to vascular endothelium.

PubMed Central

Rinker, K D; Prabhakar, V; Truskey, G A

2001-01-01

In this study we examined whether monocytic cell attachment to vascular endothelium was affected by elevating shear stress at a constant shear rate. Contact time, which is inversely related to the shear rate, was fixed and viscosity elevated with dextran to increase the shear stress (and hence the net force on the cell) independently of shear rate. At a fixed contact time, tethering frequencies increased, rolling velocities decreased, and median arrest durations increased with increasing shear stress. Rolling and short arrests (< 0.2 s) were well fit by a single exponential consistent with adhesion via the formation of a single additional bond. The cell dissociation constant, k(off), increased when the shear stress was elevated at constant shear rate. Firmly adherent cells arresting for at least 0.2 s were well fit by a stochastic model involving dissociation from multiple bonds. Therefore, at a fixed contact time and increasing shear stress, bonds formed more frequently for rolling cells resulting in more short arrests, and more bonds formed for firmly arresting cells resulting in longer arrest durations. Possible mechanisms for this increased adhesion include greater monocyte deformation and/or more frequent penetration of microvilli through steric and charge barriers. PMID:11259286

Adaptations for vision in dim light: impulse responses and bumps in nocturnal spider photoreceptor cells (Cupiennius salei Keys).

PubMed

Pirhofer-Walzl, Karin; Warrant, Eric; Barth, Friedrich G

2007-10-01

The photoreceptor cells of the nocturnal spider Cupiennius salei were investigated by intracellular electrophysiology. (1) The responses of photoreceptor cells of posterior median (PM) and anterior median (AM) eyes to short (2 ms) light pulses showed long integration times in the dark-adapted and shorter integration times in the light-adapted state. (2) At very low light intensities, the photoreceptors responded to single photons with discrete potentials, called bumps, of high amplitude (2-20 mV). When measured in profoundly dark-adapted photoreceptor cells of the PM eyes these bumps showed an integration time of 128 +/- 35 ms (n = 7) whereas in dark-adapted photoreceptor cells of AM eyes the integration time was 84 +/- 13 ms (n = 8), indicating that the AM eyes are intrinsically faster than the PM eyes. (3) Long integration times, which improve visual reliability in dim light, and large responses to single photons in the dark-adapted state, contribute to a high visual sensitivity in Cupiennius at night. This conclusion is underlined by a calculation of sensitivity that accounts for both anatomical and physiological characteristics of the eye.

Single molecule and single cell epigenomics.

PubMed

Hyun, Byung-Ryool; McElwee, John L; Soloway, Paul D

2015-01-15

Dynamically regulated changes in chromatin states are vital for normal development and can produce disease when they go awry. Accordingly, much effort has been devoted to characterizing these states under normal and pathological conditions. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is the most widely used method to characterize where in the genome transcription factors, modified histones, modified nucleotides and chromatin binding proteins are found; bisulfite sequencing (BS-seq) and its variants are commonly used to characterize the locations of DNA modifications. Though very powerful, these methods are not without limitations. Notably, they are best at characterizing one chromatin feature at a time, yet chromatin features arise and function in combination. Investigators commonly superimpose separate ChIP-seq or BS-seq datasets, and then infer where chromatin features are found together. While these inferences might be correct, they can be misleading when the chromatin source has distinct cell types, or when a given cell type exhibits any cell to cell variation in chromatin state. These ambiguities can be eliminated by robust methods that directly characterize the existence and genomic locations of combinations of chromatin features in very small inputs of cells or ideally, single cells. Here we review single molecule epigenomic methods under development to overcome these limitations, the technical challenges associated with single molecule methods and their potential application to single cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Single Molecule and Single Cell Epigenomics

PubMed Central

Hyun, Byung-Ryool; McElwee, John L.; Soloway, Paul D.

2014-01-01

Dynamically regulated changes in chromatin states are vital for normal development and can produce disease when they go awry. Accordingly, much effort has been devoted to characterizing these states under normal and pathological conditions. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is the most widely used method to characterize where in the genome transcription factors, modified histones, modified nucleotides and chromatin binding proteins are found; bisulfite sequencing (BS-seq) and its variants are commonly used to characterize the locations of DNA modifications. Though very powerful, these methods are not without limitations. Notably, they are best at characterizing one chromatin feature at a time, yet chromatin features arise and function in combination. Investigators commonly superimpose separate ChIP-seq or BS-seq datasets, and then infer where chromatin features are found together. While these inferences might be correct, they can be misleading when the chromatin source has distinct cell types, or when a given cell type exhibits any cell to cell variation in chromatin state. These ambiguities can be eliminated by robust methods that directly characterize the existence and genomic locations of combinations of chromatin features in very small inputs of cells or ideally, single cells. Here we review single molecule epigenomic methods under development to overcome these limitations, the technical challenges associated with single molecule methods and their potential application to single cells. PMID:25204781

Solid oxide fuel cell anode degradation by the effect of hydrogen chloride in stack and single cell environments

NASA Astrophysics Data System (ADS)

Madi, Hossein; Lanzini, Andrea; Papurello, Davide; Diethelm, Stefan; Ludwig, Christian; Santarelli, Massimo; Van herle, Jan

2016-09-01

The poisoning effect by hydrogen chloride (HCl) on state-of-the-art Ni anode-supported solid oxide fuel cells (SOFCs) at 750 °C is evaluated in either hydrogen or syngas fuel. Experiments are performed on single cells and short stacks and HCl concentration in the fuel gas is increased from 1 ppm(v) up to 1000 ppm(v) at different current densities. Characterization methods such as cell voltage monitoring vs. time and electrochemical impedance response analysis (distribution of relaxation times (DRT), equivalent electrical circuit) are used to identify the prevailing degradation mechanism. Single cell experiments revealed that the poisoning is more severe when feeding with hydrogen than with syngas. Performance loss is attributed to the effects of HCl adsorption onto nickel surfaces, which lowered the catalyst activity. Interestingly, in syngas HCl does not affect stack performance even at concentrations up to 500 ppm(v), even when causing severe corrosion of the anode exhaust pipe. Furthermore, post-test analysis suggests that chlorine is present on the nickel particles in the form of adsorbed chlorine, rather than forming a secondary phase of nickel chlorine.

Real-Time Single Molecule Visualization of SH2 Domain Membrane Recruitment in Growth Factor Stimulated Cells.

PubMed

Oh, Dongmyung

2017-01-01

In the last decade, single molecule tracking (SMT) techniques have emerged as a versatile tool for molecular cell biology research. This approach allows researchers to monitor the real-time behavior of individual molecules in living cells with nanometer and millisecond resolution. As a result, it is possible to visualize biological processes as they occur at a molecular level in real time. Here we describe a method for the real-time visualization of SH2 domain membrane recruitment from the cytoplasm to epidermal growth factor (EGF) induced phosphotyrosine sites on the EGF receptor. Further, we describe methods that utilize SMT data to define SH2 domain membrane dynamics parameters such as binding (τ), dissociation (k d ), and diffusion (D) rates. Together these methods may allow us to gain greater understanding of signal transduction dynamics and the molecular basis of disease-related aberrant pathways.

Stimulus encoding and feature extraction by multiple sensory neurons.

PubMed

Krahe, Rüdiger; Kreiman, Gabriel; Gabbiani, Fabrizio; Koch, Christof; Metzner, Walter

2002-03-15

Neighboring cells in topographical sensory maps may transmit similar information to the next higher level of processing. How information transmission by groups of nearby neurons compares with the performance of single cells is a very important question for understanding the functioning of the nervous system. To tackle this problem, we quantified stimulus-encoding and feature extraction performance by pairs of simultaneously recorded electrosensory pyramidal cells in the hindbrain of weakly electric fish. These cells constitute the output neurons of the first central nervous stage of electrosensory processing. Using random amplitude modulations (RAMs) of a mimic of the fish's own electric field within behaviorally relevant frequency bands, we found that pyramidal cells with overlapping receptive fields exhibit strong stimulus-induced correlations. To quantify the encoding of the RAM time course, we estimated the stimuli from simultaneously recorded spike trains and found significant improvements over single spike trains. The quality of stimulus reconstruction, however, was still inferior to the one measured for single primary sensory afferents. In an analysis of feature extraction, we found that spikes of pyramidal cell pairs coinciding within a time window of a few milliseconds performed significantly better at detecting upstrokes and downstrokes of the stimulus compared with isolated spikes and even spike bursts of single cells. Coincident spikes can thus be considered "distributed bursts." Our results suggest that stimulus encoding by primary sensory afferents is transformed into feature extraction at the next processing stage. There, stimulus-induced coincident activity can improve the extraction of behaviorally relevant features from the stimulus.

Individual motile CD4+ T cells can participate in efficient multi-killing through conjugation to multiple tumor cells

PubMed Central

Liadi, Ivan; Singh, Harjeet; Romain, Gabrielle; Rey-Villamizar, Nicolas; Merouane, Amine; Adolacion, Jay R T.; Kebriaei, Partow; Huls, Helen; Qiu, Peng; Roysam, Badrinath; Cooper, Laurence J.N.; Varadarajan, Navin

2015-01-01

T cells genetically modified to express a CD19-specific chimeric antigen receptor (CAR) for the investigational treatment of B-cell malignancies comprise a heterogeneous population, and their ability to persist and participate in serial killing of tumor cells is a predictor of therapeutic success. We implemented Timelapse Imaging Microscopy In Nanowell Grids (TIMING) to provide direct evidence that CD4+CAR+ T cells (CAR4 cells) can engage in multi-killing via simultaneous conjugation to multiple tumor cells. Comparisons of the CAR4 cells and CD8+CAR+ T cells (CAR8 cells) demonstrate that while CAR4 cells can participate in killing and multi-killing, they do so at slower rates, likely due to the lower Granzyme B content. Significantly, in both sets of T cells, a minor sub-population of individual T cells identified by their high motility, demonstrated efficient killing of single tumor cells. By comparing both the multi-killer and single killer CAR+ T cells it appears that the propensity and kinetics of T-cell apoptosis was modulated by the number of functional conjugations. T cells underwent rapid apoptosis, and at higher frequencies, when conjugated to single tumor cells in isolation and this effect was more pronounced on CAR8 cells. Our results suggest that the ability of CAR+ T cells to participate in multi-killing should be evaluated in the context of their ability to resist activation induced cell death (AICD). We anticipate that TIMING may be utilized to rapidly determine the potency of T-cell populations and may facilitate the design and manufacture of next-generation CAR+ T cells with improved efficacy. PMID:25711538

Genome-wide base-resolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing (scBS-seq).

PubMed

Clark, Stephen J; Smallwood, Sébastien A; Lee, Heather J; Krueger, Felix; Reik, Wolf; Kelsey, Gavin

2017-03-01

DNA methylation (DNAme) is an important epigenetic mark in diverse species. Our current understanding of DNAme is based on measurements from bulk cell samples, which obscures intercellular differences and prevents analyses of rare cell types. Thus, the ability to measure DNAme in single cells has the potential to make important contributions to the understanding of several key biological processes, such as embryonic development, disease progression and aging. We have recently reported a method for generating genome-wide DNAme maps from single cells, using single-cell bisulfite sequencing (scBS-seq), allowing the quantitative measurement of DNAme at up to 50% of CpG dinucleotides throughout the mouse genome. Here we present a detailed protocol for scBS-seq that includes our most recent developments to optimize recovery of CpGs, mapping efficiency and success rate; reduce hands-on time; and increase sample throughput with the option of using an automated liquid handler. We provide step-by-step instructions for each stage of the method, comprising cell lysis and bisulfite (BS) conversion, preamplification and adaptor tagging, library amplification, sequencing and, lastly, alignment and methylation calling. An individual with relevant molecular biology expertise can complete library preparation within 3 d. Subsequent computational steps require 1-3 d for someone with bioinformatics expertise.

Identification of unique release kinetics of serotonin from guinea-pig and human enterochromaffin cells

PubMed Central

Raghupathi, Ravinarayan; Duffield, Michael D; Zelkas, Leah; Meedeniya, Adrian; Brookes, Simon J H; Sia, Tiong Cheng; Wattchow, David A; Spencer, Nick J; Keating, Damien J

2013-01-01

The major source of serotonin (5-HT) in the body is the enterochromaffin (EC) cells lining the intestinal mucosa of the gastrointestinal tract. Despite the fact that EC cells synthesise ∼95% of total body 5-HT, and that this 5-HT has important paracrine and endocrine roles, no studies have investigated the mechanisms of 5-HT release from single primary EC cells. We have developed a rapid primary culture of guinea-pig and human EC cells, allowing analysis of single EC cell function using electrophysiology, electrochemistry, Ca2+ imaging, immunocytochemistry and 3D modelling. Ca2+ enters EC cells upon stimulation and triggers quantal 5-HT release via L-type Ca2+ channels. Real time amperometric techniques reveal that EC cells release 5-HT at rest and this release increases upon stimulation. Surprisingly for an endocrine cell storing 5-HT in large dense core vesicles (LDCVs), EC cells release 70 times less 5-HT per fusion event than catecholamine released from similarly sized LDCVs in endocrine chromaffin cells, and the vesicle release kinetics instead resembles that observed in mammalian synapses. Furthermore, we measured EC cell density along the gastrointestinal tract to create three-dimensional (3D) simulations of 5-HT diffusion using the minimal number of variables required to understand the physiological relevance of single cell 5-HT release in the whole-tissue milieu. These models indicate that local 5-HT levels are likely to be maintained around the activation threshold for mucosal 5-HT receptors and that this is dependent upon stimulation and location within the gastrointestinal tract. This is the first study demonstrating single cell 5-HT release in primary EC cells. The mode of 5-HT release may represent a unique mode of exocytosis amongst endocrine cells and is functionally relevant to gastrointestinal sensory and motor function. PMID:24099799

High-resolution Myogenic Lineage Mapping by Single-Cell Mass Cytometry

PubMed Central

Porpiglia, Ermelinda; Samusik, Nikolay; Ho, Andrew Tri Van; Cosgrove, Benjamin D.; Mai, Thach; Davis, Kara L.; Jager, Astraea; Nolan, Garry P.; Bendall, Sean C.; Fantl, Wendy J.; Blau, Helen M.

2017-01-01

Muscle regeneration is a dynamic process during which cell state and identity change over time. A major roadblock has been a lack of tools to resolve a myogenic progression in vivo. Here we capitalize on a transformative technology, single-cell mass cytometry (CyTOF), to identify in vivo skeletal muscle stem cell and previously unrecognized progenitor populations that precede differentiation. We discovered two cell surface markers, CD9 and CD104, whose combined expression enabled in vivo identification and prospective isolation of stem and progenitor cells. Data analysis using the X-shift algorithm paired with single-cell force directed layout visualization, defined a molecular signature of the activated stem cell state (CD44+/CD98+/MyoD+) and delineated a myogenic trajectory during recovery from acute muscle injury. Our studies uncover the dynamics of skeletal muscle regeneration in vivo and pave the way for the elucidation of the regulatory networks that underlie cell-state transitions in muscle diseases and aging. PMID:28414312

A Single-Cell Roadmap of Lineage Bifurcation in Human ESC Models of Embryonic Brain Development.

PubMed

Yao, Zizhen; Mich, John K; Ku, Sherman; Menon, Vilas; Krostag, Anne-Rachel; Martinez, Refugio A; Furchtgott, Leon; Mulholland, Heather; Bort, Susan; Fuqua, Margaret A; Gregor, Ben W; Hodge, Rebecca D; Jayabalu, Anu; May, Ryan C; Melton, Samuel; Nelson, Angelique M; Ngo, N Kiet; Shapovalova, Nadiya V; Shehata, Soraya I; Smith, Michael W; Tait, Leah J; Thompson, Carol L; Thomsen, Elliot R; Ye, Chaoyang; Glass, Ian A; Kaykas, Ajamete; Yao, Shuyuan; Phillips, John W; Grimley, Joshua S; Levi, Boaz P; Wang, Yanling; Ramanathan, Sharad

2017-01-05

During human brain development, multiple signaling pathways generate diverse cell types with varied regional identities. Here, we integrate single-cell RNA sequencing and clonal analyses to reveal lineage trees and molecular signals underlying early forebrain and mid/hindbrain cell differentiation from human embryonic stem cells (hESCs). Clustering single-cell transcriptomic data identified 41 distinct populations of progenitor, neuronal, and non-neural cells across our differentiation time course. Comparisons with primary mouse and human gene expression data demonstrated rostral and caudal progenitor and neuronal identities from early brain development. Bayesian analyses inferred a unified cell-type lineage tree that bifurcates between cortical and mid/hindbrain cell types. Two methods of clonal analyses confirmed these findings and further revealed the importance of Wnt/β-catenin signaling in controlling this lineage decision. Together, these findings provide a rich transcriptome-based lineage map for studying human brain development and modeling developmental disorders. Copyright © 2017 Elsevier Inc. All rights reserved.

Mechanics of single cells: rheology, time dependence, and fluctuations.

PubMed

Massiera, Gladys; Van Citters, Kathleen M; Biancaniello, Paul L; Crocker, John C

2007-11-15

The results of mechanical measurements on single cultured epithelial cells using both magnetic twisting cytometry (MTC) and laser tracking microrheology (LTM) are described. Our unique approach uses laser deflection for high-performance tracking of cell-adhered magnetic beads either in response to an oscillatory magnetic torque (MTC) or due to random Brownian or ATP-dependent forces (LTM). This approach is well suited for accurately determining the rheology of single cells, the study of temporal and cell-to-cell variations in the MTC signal amplitude, and assessing the statistical character of the tracers' random motion in detail. The temporal variation of the MTC rocking amplitude is surprisingly large and manifests as a frequency-independent multiplicative factor having a 1/f spectrum in living cells, which disappears upon ATP depletion. In the epithelial cells we study, random bead position fluctuations are Gaussian to the limits of detection both in the Brownian and ATP-dependent cases, unlike earlier studies on other cell types.

Shape-Dependent Optoelectronic Cell Lysis**

PubMed Central

Kremer, Clemens; Witte, Christian; Neale, Steven L; Reboud, Julien; Barrett, Michael P; Cooper, Jonathan M

2014-01-01

We show an electrical method to break open living cells amongst a population of different cell types, where cell selection is based upon their shape. We implement the technique on an optoelectronic platform, where light, focused onto a semiconductor surface from a video projector creates a reconfigurable pattern of electrodes. One can choose the area of cells to be lysed in real-time, from single cells to large areas, simply by redrawing the projected pattern. We show that the method, based on the “electrical shadow” that the cell casts, allows the detection of rare cell types in blood (including sleeping sickness parasites), and has the potential to enable single cell studies for advanced molecular diagnostics, as well as wider applications in analytical chemistry. PMID:24402800

Reusable Floating-Electrode Sensor for Real-Time Electrophysiological Monitoring of Nonadherent Cells

NASA Astrophysics Data System (ADS)

Pham Ba, Viet Anh; Ta, Van-Thao; Park, Juhun; Park, Eun Jin; Hong, Seunghun

2015-03-01

We herein report the development of a reusable floating-electrode sensor (FES) based on aligned single-walled carbon nanotubes, which allowed quantitatively monitoring the electrophysiological responses from nonadherent cells. The FES was used to measure the real-time responses of normal lung cells and small-cell lung cancer (SCLC) cells to the addition of nicotine. The SCLC cells exhibited rather large electrophysiological responses to nicotine compared to normal cells, which was attributed to the overexpressed nicotinic acetylcholine receptors (nAChRs) in the SCLC cells. Importantly, using only a single device could measure repeatedly the responses of multiple individual cells to various drugs, enabling statistically meaningful measurements without errors from the device-to-device variations of the sensor characteristics. As results, that the treatment with drugs such as genistin or daidzein reduced Ca2+ influx in SCLC cells was found. Moreover, tamoxifen, has been known as an anti-estrogen compound, was found to only partly block the binding of daidzein to nAChRs. Our FES can be a promising tool for various biomedical applications such as drug screening and therapy monitoring.

An automated image analysis framework for segmentation and division plane detection of single live Staphylococcus aureus cells which can operate at millisecond sampling time scales using bespoke Slimfield microscopy

NASA Astrophysics Data System (ADS)

Wollman, Adam J. M.; Miller, Helen; Foster, Simon; Leake, Mark C.

2016-10-01

Staphylococcus aureus is an important pathogen, giving rise to antimicrobial resistance in cell strains such as Methicillin Resistant S. aureus (MRSA). Here we report an image analysis framework for automated detection and image segmentation of cells in S. aureus cell clusters, and explicit identification of their cell division planes. We use a new combination of several existing analytical tools of image analysis to detect cellular and subcellular morphological features relevant to cell division from millisecond time scale sampled images of live pathogens at a detection precision of single molecules. We demonstrate this approach using a fluorescent reporter GFP fused to the protein EzrA that localises to a mid-cell plane during division and is involved in regulation of cell size and division. This image analysis framework presents a valuable platform from which to study candidate new antimicrobials which target the cell division machinery, but may also have more general application in detecting morphologically complex structures of fluorescently labelled proteins present in clusters of other types of cells.

LEAP: constructing gene co-expression networks for single-cell RNA-sequencing data using pseudotime ordering.

PubMed

Specht, Alicia T; Li, Jun

2017-03-01

To construct gene co-expression networks based on single-cell RNA-Sequencing data, we present an algorithm called LEAP, which utilizes the estimated pseudotime of the cells to find gene co-expression that involves time delay. R package LEAP available on CRAN. jun.li@nd.edu. Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Finite state projection based bounds to compare chemical master equation models using single-cell data

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fox, Zachary; Neuert, Gregor; Department of Pharmacology, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232

2016-08-21

Emerging techniques now allow for precise quantification of distributions of biological molecules in single cells. These rapidly advancing experimental methods have created a need for more rigorous and efficient modeling tools. Here, we derive new bounds on the likelihood that observations of single-cell, single-molecule responses come from a discrete stochastic model, posed in the form of the chemical master equation. These strict upper and lower bounds are based on a finite state projection approach, and they converge monotonically to the exact likelihood value. These bounds allow one to discriminate rigorously between models and with a minimum level of computational effort.more » In practice, these bounds can be incorporated into stochastic model identification and parameter inference routines, which improve the accuracy and efficiency of endeavors to analyze and predict single-cell behavior. We demonstrate the applicability of our approach using simulated data for three example models as well as for experimental measurements of a time-varying stochastic transcriptional response in yeast.« less

Symmetry of initial cell divisions among primitive hematopoietic progenitors is independent of ontogenic age and regulatory molecules.

PubMed

Huang, S; Law, P; Francis, K; Palsson, B O; Ho, A D

1999-10-15

We have developed a time-lapse camera system to follow the replication history and the fate of hematopoietic stem cells (HSC) at a single-cell level. Combined with single-cell culture, we correlated the early replication behavior with colony development after 14 days. The membrane dye PKH26 was used to monitor cell division. In addition to multiple, synchronous, and symmetric divisions, single-sorted CD34(+)/CD38(-) cells derived from fetal liver (FLV) also gave rise to a daughter cell that remained quiescent for up to 8 days, whereas the other daughter cell proliferated exponentially. Upon separation and replating as single cells onto medium containing a cytokine cocktail, 60.6% +/- 9.8% of the initially quiescent cells (PKH26 bright) gave rise again to colonies and 15.8% +/- 7.8% to blast colonies that could be replated. We have then determined the effects of various regulatory molecules on symmetry of initial cell divisions. After single-cell sorting, the CD34(+)/CD38(-) cells derived from FLV were exposed to flt3-ligand, thrombopoietin, stem cell factor (SCF), or medium containing a cytokine cocktail (with SCF, interleukin-3, interleukin-6, granulocyte-macrophage colony-stimulating factor, and erythropoietin). Whereas mitotic rate, colony efficiency, and asymmetric divisions could be altered using various regulatory molecules, the asymmetric division index, defined as the number of asymmetric divisions versus the number of dividing cells, was not altered significantly. This observation suggests that, although lineage commitment and cell proliferation can be skewed by extrinsic signaling, symmetry of early divisions is probably under the control of intrinsic factors.

Design of a Single-Cell Positioning Controller Using Electroosmotic Flow and Image Processing

PubMed Central

Ay, Chyung; Young, Chao-Wang; Chen, Jhong-Yin

2013-01-01

The objective of the current research was not only to provide a fast and automatic positioning platform for single cells, but also improved biomolecular manipulation techniques. In this study, an automatic platform for cell positioning using electroosmotic flow and image processing technology was designed. The platform was developed using a PCI image acquisition interface card for capturing images from a microscope and then transferring them to a computer using human-machine interface software. This software was designed by the Laboratory Virtual Instrument Engineering Workbench, a graphical language for finding cell positions and viewing the driving trace, and the fuzzy logic method for controlling the voltage or time of an electric field. After experiments on real human leukemic cells (U-937), the success of the cell positioning rate achieved by controlling the voltage factor reaches 100% within 5 s. A greater precision is obtained when controlling the time factor, whereby the success rate reaches 100% within 28 s. Advantages in both high speed and high precision are attained if these two voltage and time control methods are combined. The control speed with the combined method is about 5.18 times greater than that achieved by the time method, and the control precision with the combined method is more than five times greater than that achieved by the voltage method. PMID:23698272

Quantitative expression analysis of selected transcription factors in pavement, basal and trichome cells of mature leaves from Arabidopsis thaliana

PubMed Central

Schliep, Martin; Ebert, Berit; Simon-Rosin, Ulrike; Zoeller, Daniela

2010-01-01

Gene expression levels of several transcription factors from Arabidopsis thaliana that were described previously to be involved in leaf development and trichome formation were analysed in trichome, basal and pavement cells of mature leaves. Single cell samples of these three cells types were collected by glass micro-capillaries. Real-time reverse transcription (RT)-PCR was used to analyse expression patterns of the following transcription factors: MYB23, MYB55, AtHB1, FILAMENTOUS FLOWER (FIL)/YABBY1 (YAB1), TRIPTYCHON (TRY) and CAPRICE (CPC). A difference in the expression patterns of TRY and CPC was revealed. Contrary to the CPC expression pattern, no transcripts of TRY could be detected in pavement cells. FIL/YAB1 was exclusively expressed in trichome cells. AtHB1 was highly expressed throughout all three cell types. MYB55 was higher expressed in basal cells than in trichome and pavement cells. MYB23 showed a pattern of low expression in pavement cells, medium in basal cells and high expression in trichomes. Expression patterns obtained by single cell sampling and real-time RT-PCR were compared to promoter GUS fusions of the selected transcription factors. Therefore, we regenerated two transgenic Arabidopsis lines that expressed the GUS reporter gene under control of the promoters of MYB55 and YAB1. In conclusion, despite their function in leaf morphogenesis, all six transcription factors were detected in mature leaves. Furthermore, single cell sampling and promoter GUS staining patterns demonstrated the predominant presence of MYB55 in basal cells as compared to pavement cells and trichomes. PMID:20101514

Quantitative expression analysis of selected transcription factors in pavement, basal and trichome cells of mature leaves from Arabidopsis thaliana.

PubMed

Schliep, Martin; Ebert, Berit; Simon-Rosin, Ulrike; Zoeller, Daniela; Fisahn, Joachim

2010-05-01

Gene expression levels of several transcription factors from Arabidopsis thaliana that were described previously to be involved in leaf development and trichome formation were analysed in trichome, basal and pavement cells of mature leaves. Single cell samples of these three cells types were collected by glass micro-capillaries. Real-time reverse transcription (RT)-PCR was used to analyse expression patterns of the following transcription factors: MYB23, MYB55, AtHB1, FILAMENTOUS FLOWER (FIL)/YABBY1 (YAB1), TRIPTYCHON (TRY) and CAPRICE (CPC). A difference in the expression patterns of TRY and CPC was revealed. Contrary to the CPC expression pattern, no transcripts of TRY could be detected in pavement cells. FIL/YAB1 was exclusively expressed in trichome cells. AtHB1 was highly expressed throughout all three cell types. MYB55 was higher expressed in basal cells than in trichome and pavement cells. MYB23 showed a pattern of low expression in pavement cells, medium in basal cells and high expression in trichomes. Expression patterns obtained by single cell sampling and real-time RT-PCR were compared to promoter GUS fusions of the selected transcription factors. Therefore, we regenerated two transgenic Arabidopsis lines that expressed the GUS reporter gene under control of the promoters of MYB55 and YAB1. In conclusion, despite their function in leaf morphogenesis, all six transcription factors were detected in mature leaves. Furthermore, single cell sampling and promoter GUS staining patterns demonstrated the predominant presence of MYB55 in basal cells as compared to pavement cells and trichomes.

Magnetophoretic circuits for digital control of single particles and cells

NASA Astrophysics Data System (ADS)

Lim, Byeonghwa; Reddy, Venu; Hu, Xinghao; Kim, Kunwoo; Jadhav, Mital; Abedini-Nassab, Roozbeh; Noh, Young-Woock; Lim, Yong Taik; Yellen, Benjamin B.; Kim, Cheolgi

2014-05-01

The ability to manipulate small fluid droplets, colloidal particles and single cells with the precision and parallelization of modern-day computer hardware has profound applications for biochemical detection, gene sequencing, chemical synthesis and highly parallel analysis of single cells. Drawing inspiration from general circuit theory and magnetic bubble technology, here we demonstrate a class of integrated circuits for executing sequential and parallel, timed operations on an ensemble of single particles and cells. The integrated circuits are constructed from lithographically defined, overlaid patterns of magnetic film and current lines. The magnetic patterns passively control particles similar to electrical conductors, diodes and capacitors. The current lines actively switch particles between different tracks similar to gated electrical transistors. When combined into arrays and driven by a rotating magnetic field clock, these integrated circuits have general multiplexing properties and enable the precise control of magnetizable objects.

Early dynamic fate changes in haemogenic endothelium characterized at the single-cell level

NASA Astrophysics Data System (ADS)

Swiers, Gemma; Baumann, Claudia; O'Rourke, John; Giannoulatou, Eleni; Taylor, Stephen; Joshi, Anagha; Moignard, Victoria; Pina, Cristina; Bee, Thomas; Kokkaliaris, Konstantinos D.; Yoshimoto, Momoko; Yoder, Mervin C.; Frampton, Jon; Schroeder, Timm; Enver, Tariq; Göttgens, Berthold; de Bruijn, Marella F. T. R.

2013-12-01

Haematopoietic stem cells (HSCs) are the founding cells of the adult haematopoietic system, born during ontogeny from a specialized subset of endothelium, the haemogenic endothelium (HE) via an endothelial-to-haematopoietic transition (EHT). Although recently imaged in real time, the underlying mechanism of EHT is still poorly understood. We have generated a Runx1 +23 enhancer-reporter transgenic mouse (23GFP) for the prospective isolation of HE throughout embryonic development. Here we perform functional analysis of over 1,800 and transcriptional analysis of 268 single 23GFP+ HE cells to explore the onset of EHT at the single-cell level. We show that initiation of the haematopoietic programme occurs in cells still embedded in the endothelial layer, and is accompanied by a previously unrecognized early loss of endothelial potential before HSCs emerge. Our data therefore provide important insights on the timeline of early haematopoietic commitment.

Single-cell analysis of HIV-1 transcriptional activity reveals expression of proviruses in expanded clones during ART

PubMed Central

Wiegand, Ann; Spindler, Jonathan; Hong, Feiyu F.; Shao, Wei; Cyktor, Joshua C.; Cillo, Anthony R.; Halvas, Elias K.; Coffin, John M.; Mellors, John W.; Kearney, Mary F.

2017-01-01

Little is known about the fraction of human immunodeficiency virus type 1 (HIV-1) proviruses that express unspliced viral RNA in vivo or about the levels of HIV RNA expression within single infected cells. We developed a sensitive cell-associated HIV RNA and DNA single-genome sequencing (CARD-SGS) method to investigate fractional proviral expression of HIV RNA (1.3-kb fragment of p6, protease, and reverse transcriptase) and the levels of HIV RNA in single HIV-infected cells from blood samples obtained from individuals with viremia or individuals on long-term suppressive antiretroviral therapy (ART). Spiking experiments show that the CARD-SGS method can detect a single cell expressing HIV RNA. Applying CARD-SGS to blood mononuclear cells in six samples from four HIV-infected donors (one with viremia and not on ART and three with viremia suppressed on ART) revealed that an average of 7% of proviruses (range: 2–18%) expressed HIV RNA. Levels of expression varied from one to 62 HIV RNA molecules per cell (median of 1). CARD-SGS also revealed the frequent expression of identical HIV RNA sequences across multiple single cells and across multiple time points in donors on suppressive ART consistent with constitutive expression of HIV RNA in infected cell clones. Defective proviruses were found to express HIV RNA at levels similar to those proviruses that had no obvious defects. CARD-SGS is a useful tool to characterize fractional proviral expression in single infected cells that persist despite ART and to assess the impact of experimental interventions on proviral populations and their expression. PMID:28416661

Human T cells monitored by impedance spectrometry using field-effect transistor arrays: a novel tool for single-cell adhesion and migration studies.

PubMed

Law, Jessica Ka Yan; Susloparova, Anna; Vu, Xuan Thang; Zhou, Xiao; Hempel, Felix; Qu, Bin; Hoth, Markus; Ingebrandt, Sven

2015-05-15

Cytotoxic T lymphocytes (CTLs) play an important role in the immune system by recognizing and eliminating pathogen-infected and tumorigenic cells. In order to achieve their function, T cells have to migrate throughout the whole body and identify the respective targets. In conventional immunology studies, interactions between CTLs and targets are usually investigated using tedious and time-consuming immunofluorescence imaging. However, there is currently no straightforward measurement tool available to examine the interaction strengths. In the present study, adhesion strengths and migration of single human CD8(+) T cells on pre-coated field-effect transistor (FET) devices (i.e. fibronectin, anti-CD3 antibody, and anti-LFA-1 antibody) were measured using impedance spectroscopy. Adhesion strengths to different protein and antibody coatings were compared. By fitting the data to an electronically equivalent circuit model, cell-related parameters (cell membrane capacitance referring to cell morphology and seal resistance referring to adhesion strength) were obtained. This electronically-assessed adhesion strength provides a novel, fast, and important index describing the interaction efficiency. Furthermore, the size of our detection transistor gates as well as their sensitivity reaches down to single cell resolution. Real-time motions of individually migrating T cells can be traced using our FET devices. The in-house fabricated FETs used in the present study are providing a novel and very efficient insight to individual cell interactions. Copyright © 2014 Elsevier B.V. All rights reserved.

Concurrent Isolation of 3 Distinct Cardiac Stem Cell Populations From a Single Human Heart Biopsy.

PubMed

Monsanto, Megan M; White, Kevin S; Kim, Taeyong; Wang, Bingyan J; Fisher, Kristina; Ilves, Kelli; Khalafalla, Farid G; Casillas, Alexandria; Broughton, Kathleen; Mohsin, Sadia; Dembitsky, Walter P; Sussman, Mark A

2017-07-07

The relative actions and synergism between distinct myocardial-derived stem cell populations remain obscure. Ongoing debates on optimal cell population(s) for treatment of heart failure prompted implementation of a protocol for isolation of multiple stem cell populations from a single myocardial tissue sample to develop new insights for achieving myocardial regeneration. Establish a robust cardiac stem cell isolation and culture protocol to consistently generate 3 distinct stem cell populations from a single human heart biopsy. Isolation of 3 endogenous cardiac stem cell populations was performed from human heart samples routinely discarded during implantation of a left ventricular assist device. Tissue explants were mechanically minced into 1 mm 3 pieces to minimize time exposure to collagenase digestion and preserve cell viability. Centrifugation removes large cardiomyocytes and tissue debris producing a single cell suspension that is sorted using magnetic-activated cell sorting technology. Initial sorting is based on tyrosine-protein kinase Kit (c-Kit) expression that enriches for 2 c-Kit + cell populations yielding a mixture of cardiac progenitor cells and endothelial progenitor cells. Flowthrough c-Kit - mesenchymal stem cells are positively selected by surface expression of markers CD90 and CD105. After 1 week of culture, the c-Kit + population is further enriched by selection for a CD133 + endothelial progenitor cell population. Persistence of respective cell surface markers in vitro is confirmed both by flow cytometry and immunocytochemistry. Three distinct cardiac cell populations with individualized phenotypic properties consistent with cardiac progenitor cells, endothelial progenitor cells, and mesenchymal stem cells can be successfully concurrently isolated and expanded from a single tissue sample derived from human heart failure patients. © 2017 American Heart Association, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brechenmacher, Laurent; Nguyen, Tran H.; Hixson, Kim K.

Root hairs are a terminally differentiated single cell type, mainly involved in water and nutrient uptake from the soil. The soybean root hair cell represents an excellent model for the study of single cell systems biology. In this study, we identified 5702 proteins, with at least two peptides, from soybean root hairs using an accurate mass and time tag approach, establishing the most comprehensive proteome reference map of this single cell type. We also showed that trypsin is the most appropriate enzyme for soybean proteomic studies by performing an in silico digestion of the soybean proteome database using different proteases.more » Although the majority of proteins identified in this study are involved in basal metabolism, the function of others are more related to root hair formation/function and include proteins involved in nutrient uptake (transporters) or vesicular trafficking (cytoskeleton and RAB proteins). Interestingly, some of these proteins appear to be specifically expressed in root hairs and constitute very good candidates for further studies to elucidate unique features of this single cell model.« less

Assessment of the effect of a Salmonella enterica ser. Typhimurium culture supernatant on the single-cell lag time of foodborne pathogens.

PubMed

Blana, Vasiliki A; Lianou, Alexandra; Nychas, George-John E

2015-12-23

The objective of this study was the in vitro evaluation of the effect of a cell-free microbial supernatant, produced by a luxS-positive Salmonella enterica ser. Typhimurium strain, on the single-cell growth kinetic behavior of two strains of S. enterica (serotypes Enteritidis and Typhimurium) and a methicillin-resistant Staphylococcus aureus strain. The single-cell lag time (λ) of the pathogens was estimated in the absence and presence (20% v/v) of microbial supernatant based on optical density measurements. As demonstrated by the obtained results, the tested microbial supernatant had a strain-specific effect on the single-cell λ and its variability. Although the mean λ values were similar in the absence and presence of microbial supernatant in the case of Salmonella Enteritidis, a significant (P ≤ 0.05) reduction and increase in the mean value of this parameter in the presence of microbial supernatant were observed for Salmonella Typhimurium and St. aureus, respectively. With regard to the effect of the tested microbial supernatant on the single-cell variability of λ, similar λ distributions were obtained in its absence and presence for S. Enteritidis, while considerable differences were noted for the other two tested organisms; the coefficient of variation of λ in the absence and presence of microbial supernatant was 41.6 and 69.8% for S. Typhimurium, respectively, with the corresponding values for St. aureus being 74.0 and 56.9%. As demonstrated by the results of bioassays, the tested microbial supernatant exhibited autoinducer-2 activity, indicating a potential association of such quorum sensing compounds with the observed effects. Although preliminary in nature, the collected data provide a good basis for future research on the role of quorum sensing in the single-cell growth behavior of foodborne pathogens.

Thermal evaluation of laser exposures in an in vitro retinal model by microthermal sensing

NASA Astrophysics Data System (ADS)

Choi, Tae Y.; Denton, Michael L.; Noojin, Gary D.; Estlack, Larry E.; Shrestha, Ramesh; Rockwell, Benjamin A.; Thomas, Robert; Kim, Dongsik

2014-09-01

A temperature detection system using a micropipette thermocouple sensor was developed for use within mammalian cells during laser exposure with an 8.6-μm beam at 532 nm. We have demonstrated the capability of measuring temperatures at a single-cell level in the microscale range by inserting micropipette-based thermal sensors of size ranging from 2 to 4 μm into the membrane of a live retinal pigment epithelium (RPE) cell subjected to a laser beam. We setup the treatment groups of 532-nm laser-irradiated single RPE cell and in situ temperature recordings were made over time. Thermal profiles are given for representative cells experiencing damage resulting from exposures of 0.2 to 2 s. The measured maximum temperature rise for each cell ranges from 39 to 73°C the RPE cells showed a signature of death for all the cases reported herein. In order to check the cell viability, real-time fluorescence microscopy was used to identify the transition of pigmented RPE cells between viable and damaged states due to laser exposure.

Motility-Driven Glass and Jamming Transitions in Biological Tissues

NASA Astrophysics Data System (ADS)

Bi, Dapeng; Yang, Xingbo; Marchetti, M. Cristina; Manning, M. Lisa

2016-04-01

Cell motion inside dense tissues governs many biological processes, including embryonic development and cancer metastasis, and recent experiments suggest that these tissues exhibit collective glassy behavior. To make quantitative predictions about glass transitions in tissues, we study a self-propelled Voronoi model that simultaneously captures polarized cell motility and multibody cell-cell interactions in a confluent tissue, where there are no gaps between cells. We demonstrate that the model exhibits a jamming transition from a solidlike state to a fluidlike state that is controlled by three parameters: the single-cell motile speed, the persistence time of single-cell tracks, and a target shape index that characterizes the competition between cell-cell adhesion and cortical tension. In contrast to traditional particulate glasses, we are able to identify an experimentally accessible structural order parameter that specifies the entire jamming surface as a function of model parameters. We demonstrate that a continuum soft glassy rheology model precisely captures this transition in the limit of small persistence times and explain how it fails in the limit of large persistence times. These results provide a framework for understanding the collective solid-to-liquid transitions that have been observed in embryonic development and cancer progression, which may be associated with epithelial-to-mesenchymal transition in these tissues.

Fluorescence from a single Symbiodinium cell

NASA Astrophysics Data System (ADS)

Guzman, Christine; Han, Xue; Shoguchi, Eiichi; Chormaic, Síle Nic

2018-07-01

The partnership between coral and its algal symbionts, Symbiodinium, is crucial to the global environment. Yet, the regulatory process within the photosynthetic machinery of Symbiodinium is still not clearly understood. Here, we studied the influence of light stress from focussed red and blue lasers on single Symbiodinium cells. Fluorescence signals were measured to show cell response. Increasing the incident laser power or the exposure time resulted in an increase followed by a decline in fluorescence intensity. The trend of fluorescence intensity changes was associated with mechanisms of light use efficiency, non-photochemical quenching, photoinhibition, and repair of the cell. Our study provides new approaches to studying the photobiology and physiology of Symbiodinium cells.

Proton sensitivity of rat cerebellar granule cell GABAA receptors: dependence on neuronal development

PubMed Central

Krishek, Belinda J; Smart, Trevor G

2001-01-01

The effect of GABAA receptor development in culture on the modulation of GABA-induced currents by external H+ was examined in cerebellar granule cells using whole-cell and single-channel recording. Equilibrium concentration-response curves revealed a lower potency for GABA between 11 and 12 days in vitro (DIV) resulting in a shift of the EC50 from 10.7 to 2.4 μM. For granule cells before 11 DIV, the peak GABA-activated current was inhibited at low external pH and enhanced at high pH with a pKa of 6.65. For the steady-state response, low pH was inhibitory with a pKa of 5.56. After 11 DIV, the peak GABA-activated current was largely pH insensitive; however, the steady-state current was potentiated at low pH with a pKa of 6.84. Single GABA-activated ion channels were recorded from outside-out patches of granule cell bodies. At pH 5.4-9.4, single GABA channels exhibited multiple conductance states occurring at 22-26, 16-17 and 12-14 pS. The conductance levels were not significantly altered over the time period of study, nor by changing the external H+ concentration. Two exponential functions were required to fit the open-time frequency histograms at both early (< 11 DIV) and late (> 11 DIV) development times at each H+ concentration. The short and long open time constants were unaffected either by the extracellular H+ concentration or by neuronal development. The distribution of all shut times was fitted by the sum of three exponentials designated as short, intermediate and long. At acidic pH, the long shut time constant decreased with development as did the relative contribution of these components to the overall distribution. This was concurrent with an increase in the mean probability of channel opening. In conclusion, this study demonstrates in cerebellar granule cells that external pH can either reduce, have no effect on, or enhance GABA-activated responses depending on the stage of development, possibly related to the subunit composition of the GABAA receptors. The mode of interaction of H+ at the single-channel level and implications of such interactions at cerebellar granule cell GABAA receptors are discussed. PMID:11208970

3D+time acquisitions of 3D cell culture by means of lens-free tomographic microscopy

NASA Astrophysics Data System (ADS)

Berdeu, Anthony; Laperrousaz, Bastien; Bordy, Thomas; Morales, S.; Gidrol, Xavier; Picollet-D'hahan, Nathalie; Allier, Cédric

2018-02-01

We propose a three-dimensional (3D) imaging platform based on lens-free microscopy to perform multi-angle acquisitions on 3D cell cultures embedded in extracellular matrix (ECM). We developed algorithms based on the Fourier diffraction theorem to perform fully 3D reconstructions of biological samples and we adapted the lens-free microscope to incubator conditions. Here we demonstrate for the first time, 3D+time lens-free acquisitions of 3D cell culture over 8 days directly into the incubator. The 3D reconstructed volume is as large as 5 mm3 and provides a unique way to observe in the same 3D cell culture experiment multiple cell migration strategies. Namely, in a 3D cell culture of prostate epithelial cells embedded within a Matrigel® matrix, we are able to distinguish single cell 'leaders', migration of cell clusters, migration of large aggregates of cells, and also close-gap and large-scale branching. In addition, we observe long-scale 3D deformations of the ECM that modify the geometry of the 3D cell culture. Interestingly, we also observed the opposite, i.e. we found that large aggregates of cells may deform the ECM by generating traction forces over very long distances. In sum we put forward a novel 3D lens-free microscopy tomographic technique to study the single and collective cell migrations, the cell-to-cell interactions and the cell-to-matrix interactions.

Spatial modeling of the membrane-cytosolic interface in protein kinase signal transduction

PubMed Central

Schröder, Andreas

2018-01-01

The spatial architecture of signaling pathways and the interaction with cell size and morphology are complex, but little understood. With the advances of single cell imaging and single cell biology, it becomes crucial to understand intracellular processes in time and space. Activation of cell surface receptors often triggers a signaling cascade including the activation of membrane-attached and cytosolic signaling components, which eventually transmit the signal to the cell nucleus. Signaling proteins can form steep gradients in the cytosol, which cause strong cell size dependence. We show that the kinetics at the membrane-cytosolic interface and the ratio of cell membrane area to the enclosed cytosolic volume change the behavior of signaling cascades significantly. We suggest an estimate of average concentration for arbitrary cell shapes depending on the cell volume and cell surface area. The normalized variance, known from image analysis, is suggested as an alternative measure to quantify the deviation from the average concentration. A mathematical analysis of signal transduction in time and space is presented, providing analytical solutions for different spatial arrangements of linear signaling cascades. Quantification of signaling time scales reveals that signal propagation is faster at the membrane than at the nucleus, while this time difference decreases with the number of signaling components in the cytosol. Our investigations are complemented by numerical simulations of non-linear cascades with feedback and asymmetric cell shapes. We conclude that intracellular signal propagation is highly dependent on cell geometry and, thereby, conveys information on cell size and shape to the nucleus. PMID:29630597

Single-Cell-Based Analysis Highlights a Surge in Cell-to-Cell Molecular Variability Preceding Irreversible Commitment in a Differentiation Process

PubMed Central

Boullu, Loïs; Morin, Valérie; Vallin, Elodie; Guillemin, Anissa; Papili Gao, Nan; Cosette, Jérémie; Arnaud, Ophélie; Kupiec, Jean-Jacques; Espinasse, Thibault

2016-01-01

In some recent studies, a view emerged that stochastic dynamics governing the switching of cells from one differentiation state to another could be characterized by a peak in gene expression variability at the point of fate commitment. We have tested this hypothesis at the single-cell level by analyzing primary chicken erythroid progenitors through their differentiation process and measuring the expression of selected genes at six sequential time-points after induction of differentiation. In contrast to population-based expression data, single-cell gene expression data revealed a high cell-to-cell variability, which was masked by averaging. We were able to show that the correlation network was a very dynamical entity and that a subgroup of genes tend to follow the predictions from the dynamical network biomarker (DNB) theory. In addition, we also identified a small group of functionally related genes encoding proteins involved in sterol synthesis that could act as the initial drivers of the differentiation. In order to assess quantitatively the cell-to-cell variability in gene expression and its evolution in time, we used Shannon entropy as a measure of the heterogeneity. Entropy values showed a significant increase in the first 8 h of the differentiation process, reaching a peak between 8 and 24 h, before decreasing to significantly lower values. Moreover, we observed that the previous point of maximum entropy precedes two paramount key points: an irreversible commitment to differentiation between 24 and 48 h followed by a significant increase in cell size variability at 48 h. In conclusion, when analyzed at the single cell level, the differentiation process looks very different from its classical population average view. New observables (like entropy) can be computed, the behavior of which is fully compatible with the idea that differentiation is not a “simple” program that all cells execute identically but results from the dynamical behavior of the underlying molecular network. PMID:28027290

Single-Cell-Based Analysis Highlights a Surge in Cell-to-Cell Molecular Variability Preceding Irreversible Commitment in a Differentiation Process.

PubMed

Richard, Angélique; Boullu, Loïs; Herbach, Ulysse; Bonnafoux, Arnaud; Morin, Valérie; Vallin, Elodie; Guillemin, Anissa; Papili Gao, Nan; Gunawan, Rudiyanto; Cosette, Jérémie; Arnaud, Ophélie; Kupiec, Jean-Jacques; Espinasse, Thibault; Gonin-Giraud, Sandrine; Gandrillon, Olivier

2016-12-01

In some recent studies, a view emerged that stochastic dynamics governing the switching of cells from one differentiation state to another could be characterized by a peak in gene expression variability at the point of fate commitment. We have tested this hypothesis at the single-cell level by analyzing primary chicken erythroid progenitors through their differentiation process and measuring the expression of selected genes at six sequential time-points after induction of differentiation. In contrast to population-based expression data, single-cell gene expression data revealed a high cell-to-cell variability, which was masked by averaging. We were able to show that the correlation network was a very dynamical entity and that a subgroup of genes tend to follow the predictions from the dynamical network biomarker (DNB) theory. In addition, we also identified a small group of functionally related genes encoding proteins involved in sterol synthesis that could act as the initial drivers of the differentiation. In order to assess quantitatively the cell-to-cell variability in gene expression and its evolution in time, we used Shannon entropy as a measure of the heterogeneity. Entropy values showed a significant increase in the first 8 h of the differentiation process, reaching a peak between 8 and 24 h, before decreasing to significantly lower values. Moreover, we observed that the previous point of maximum entropy precedes two paramount key points: an irreversible commitment to differentiation between 24 and 48 h followed by a significant increase in cell size variability at 48 h. In conclusion, when analyzed at the single cell level, the differentiation process looks very different from its classical population average view. New observables (like entropy) can be computed, the behavior of which is fully compatible with the idea that differentiation is not a "simple" program that all cells execute identically but results from the dynamical behavior of the underlying molecular network.

Just-in-time vaccines: Biomineralized calcium phosphate core-immunogen shell nanoparticles induce long-lasting CD8(+) T cell responses in mice.

PubMed

Zhou, Weibin; Moguche, Albanus O; Chiu, David; Murali-Krishna, Kaja; Baneyx, François

2014-04-01

Distributed and on-demand vaccine production could be game-changing for infectious disease treatment in the developing world by providing new therapeutic opportunities and breaking the refrigeration "cold chain". Here, we show that a fusion protein between a calcium phosphate binding domain and the model antigen ovalbumin can mineralize a biocompatible adjuvant in a single step. The resulting 50 nm calcium phosphate core-immunogen shell particles are comparable to soluble protein in inducing ovalbumin-specific antibody response and class switch recombination in mice. However, single dose vaccination with nanoparticles leads to higher expansion of ovalbumin-specific CD8(+) T cells upon challenge with an influenza virus bearing the ovalbumin-derived SIINFEKL peptide, and these cells produce high levels of IFN-γ. Furthermore, mice exhibit a robust antigen-specific CD8(+) T cell recall response when challenged with virus 8 months post-immunization. These results underscore the promise of immunogen-controlled adjuvant mineralization for just-in-time manufacturing of effective T cell vaccines. This paper reports that a fusion protein between a calcium phosphate binding domain and the model antigen ovalbumin can mineralize into a biocompatible adjuvant in a single step, enabling distributed and on-demand vaccine production and eliminating the need for refrigeration of vaccines. The findings highlight the possibility of immunogen-controlled adjuvant mineralization for just-in-time manufacturing of effective T cell vaccines. Copyright © 2014 Elsevier Inc. All rights reserved.

Cavity ring-down spectroscopy in the liquid phase

NASA Astrophysics Data System (ADS)

Xu, Shucheng; Sha, Guohe; Xie, Jinchun

2002-02-01

A new application for cavity ring-down spectroscopic (CRDS) technique using a pulsed polarized light source has been developed in the absorption measurement of liquids for "colorless" organic compounds using both a single sample cell and double sample cells inserted in an optical cavity at Brewster angle. At present an experimental capability of measuring absorption coefficients as small as 2-5×10-7 cm-1 has been demonstrated by measurement of the absorption baselines. The first spectra for CRDS in the liquid phase, the C-H stretching fifth vibrational overtones of benzene in the pure liquid and hexane solution are obtained. The optical absorption length for liquids in both a single sample cell and double sample cells of 1 cm length is up to 900 cm due to multipass of light within an optical cavity. Compared to the thermal lens and optoacoustic spectroscopic techniques, the sensitivity for CRDS mainly depends on the optical absorption path of the sample (single passing path of the sample times multipass times), is not determined by the laser power and the length of the sample cell. The absolute absorption coefficient and band intensity for the sample are determined directly by the spectroscopy.

Size- and time-dependent growth properties of human induced pluripotent stem cells in the culture of single aggregate.

PubMed

Nath, Suman C; Horie, Masanobu; Nagamori, Eiji; Kino-Oka, Masahiro

2017-10-01

Aggregate culture of human induced pluripotent stem cells (hiPSCs) is a promising method to obtain high number of cells for cell therapy applications. This study quantitatively evaluated the effects of initial cell number and culture time on the growth of hiPSCs in the culture of single aggregate. Small size aggregates ((1.1 ± 0.4) × 10 1 -(2.8 ± 0.5) × 10 1 cells/aggregate) showed a lower growth rate in comparison to medium size aggregates ((8.8 ± 0.8) × 10 1 -(6.8 ± 1.1) × 10 2 cells/aggregate) during early-stage of culture (24-72 h). However, when small size aggregates were cultured in conditioned medium, their growth rate increased significantly. On the other hand, large size aggregates ((1.1 ± 0.2) × 10 3 -(3.5 ± 1.1) × 10 3 cells/aggregate) showed a lower growth rate and lower expression level of proliferation marker (ki-67) in the center region of aggregate in comparison to medium size aggregate during early-stage of culture. Medium size aggregates showed the highest growth rate during early-stage of culture. Furthermore, hiPSCs proliferation was dependent on culture time because the growth rate decreased significantly during late-stage of culture (72-120 h) at which point collagen type I accumulated on the periphery of aggregate, suggesting blockage of diffusive transport of nutrients, oxygen and metabolites into and out of the aggregates. Consideration of initial cell number and culture time are important to maintain balance between autocrine factors secretion and extracellular matrix accumulation on the aggregate periphery to achieve optimal growth of hiPSCs in the culture of single aggregate. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Single α-particle irradiation permits real-time visualization of RNF8 accumulation at DNA damaged sites

NASA Astrophysics Data System (ADS)

Muggiolu, Giovanna; Pomorski, Michal; Claverie, Gérard; Berthet, Guillaume; Mer-Calfati, Christine; Saada, Samuel; Devès, Guillaume; Simon, Marina; Seznec, Hervé; Barberet, Philippe

2017-01-01

As well as being a significant source of environmental radiation exposure, α-particles are increasingly considered for use in targeted radiation therapy. A better understanding of α-particle induced damage at the DNA scale can be achieved by following their tracks in real-time in targeted living cells. Focused α-particle microbeams can facilitate this but, due to their low energy (up to a few MeV) and limited range, α-particles detection, delivery, and follow-up observations of radiation-induced damage remain difficult. In this study, we developed a thin Boron-doped Nano-Crystalline Diamond membrane that allows reliable single α-particles detection and single cell irradiation with negligible beam scattering. The radiation-induced responses of single 3 MeV α-particles delivered with focused microbeam are visualized in situ over thirty minutes after irradiation by the accumulation of the GFP-tagged RNF8 protein at DNA damaged sites.

Auto-luminescent genetically encoded ratiometric indicator for real-time Ca2+ imaging at the single cell level

NASA Astrophysics Data System (ADS)

Saito, Kenta; Kobayashi, Kentaro; Nagai, Takeharu

2011-12-01

Efficient bioluminescence resonance energy transfer (BRET) from a bioluminescent protein to a fluorescent protein with high fluorescent quantum yield has been utilized to enhance luminescence intensity, allowing single-cell imaging in near real time without external light illumination. We have applied this strategy to develop an autoluminescent Ca2+ indicator, BRAC, which is composed of Ca2+-binding protein, calmodulin, and its target peptide, M13, sandwiched between a yellow fluorescent protein variant, Venus, and an enhanced Renilla luciferase, RLuc8. With this BRAC, we succeeded visualization of Ca2+ dynamics at the single-cell level with temporal resolution at 1 Hz. Moreover, BRAC signals were acquired by ratiometric imaging capable of canceling out Ca2+-independent signal drifts due to change in cell shape, focus shift, etc. Taking advantage of the bioluminescence imaging property that does not require external excitation light, BRAC might become a powerful tool applicable in conjunction with so-called optogenetic technology by which we can control cellular and protein function by light illumination.

Analysis of mitosis and antimitotic drug responses in tumors by in vivo microscopy and single-cell pharmacodynamics.

PubMed

Orth, James D; Kohler, Rainer H; Foijer, Floris; Sorger, Peter K; Weissleder, Ralph; Mitchison, Timothy J

2011-07-01

Cancer relies upon frequent or abnormal cell division, but how the tumor microenvironment affects mitotic processes in vivo remains unclear, largely due to the technical challenges of optical access, spatial resolution, and motion. We developed high-resolution in vivo microscopy methods to visualize mitosis in a murine xenograft model of human cancer. Using these methods, we determined whether the single-cell response to the antimitotic drug paclitaxel (Ptx) was the same in tumors as in cell culture, observed the impact of Ptx on the tumor response as a whole, and evaluated the single-cell pharmacodynamics (PD) of Ptx (by in vivo PD microscopy). Mitotic initiation was generally less frequent in tumors than in cell culture, but subsequently it proceeded normally. Ptx treatment caused spindle assembly defects and mitotic arrest, followed by slippage from mitotic arrest, multinucleation, and apoptosis. Compared with cell culture, the peak mitotic index in tumors exposed to Ptx was lower and the tumor cells survived longer after mitotic arrest, becoming multinucleated rather than dying directly from mitotic arrest. Thus, the tumor microenvironment was much less proapoptotic than cell culture. The morphologies associated with mitotic arrest were dose and time dependent, thereby providing a semiquantitative, single-cell measure of PD. Although many tumor cells did not progress through Ptx-induced mitotic arrest, tumor significantly regressed in the model. Our findings show that in vivo microscopy offers a useful tool to visualize mitosis during tumor progression, drug responses, and cell fate at the single-cell level. ©2011 AACR.

Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy

PubMed Central

Young, Jonathan W; Locke, James C W; Altinok, Alphan; Rosenfeld, Nitzan; Bacarian, Tigran; Swain, Peter S; Mjolsness, Eric; Elowitz, Michael B

2014-01-01

Quantitative single-cell time-lapse microscopy is a powerful method for analyzing gene circuit dynamics and heterogeneous cell behavior. We describe the application of this method to imaging bacteria by using an automated microscopy system. This protocol has been used to analyze sporulation and competence differentiation in Bacillus subtilis, and to quantify gene regulation and its fluctuations in individual Escherichia coli cells. The protocol involves seeding and growing bacteria on small agarose pads and imaging the resulting microcolonies. Images are then reviewed and analyzed using our laboratory's custom MATLAB analysis code, which segments and tracks cells in a frame-to-frame method. This process yields quantitative expression data on cell lineages, which can illustrate dynamic expression profiles and facilitate mathematical models of gene circuits. With fast-growing bacteria, such as E. coli or B. subtilis, image acquisition can be completed in 1 d, with an additional 1–2 d for progressing through the analysis procedure. PMID:22179594

Water Triple-Point Comparisons: Plateau Averaging or Peak Value?

NASA Astrophysics Data System (ADS)

Steur, P. P. M.; Dematteis, R.

2014-04-01

With a certain regularity, national metrology institutes conduct comparisons of water triple-point (WTP) cells. The WTP is the most important fixed point for the International Temperature Scale of 1990 (ITS-90). In such comparisons, it is common practice to simply average all the single measured temperature points obtained on a single ice mantle. This practice is quite reasonable whenever the measurements show no time dependence in the results. Ever since the first Supplementary Information for the International Temperature Scale of 1990, published by the Bureau International des Poids et Mesures in Sèvres, it was strongly suggested to wait at least 1 day before taking measurements (now up to 10 days), in order for a newly created ice mantle to stabilize. This stabilization is accompanied by a change in temperature with time. A recent improvement in the sensitivity of resistance measurement enabled the Istituto Nazionale di Ricerca Metrologica to detect more clearly the (possible) change in temperature with time of the WTP on a single ice mantle, as for old borosilicate cells. A limited investigation was performed where the temperature of two cells was monitored day-by-day, from the moment of mantle creation, where it was found that with (old) borosilicate cells it may be counterproductive to wait the usual week before starting measurements. The results are presented and discussed, and it is suggested to adapt the standard procedure for comparisons of WTP cells allowing for a different data treatment with (old) borosilicate cells, because taking the temperature dependence into account will surely reduce the reported differences between cells.

Advances in single-cell experimental design made possible by automated imaging platforms with feedback through segmentation.

PubMed

Crick, Alex J; Cammarota, Eugenia; Moulang, Katie; Kotar, Jurij; Cicuta, Pietro

2015-01-01

Live optical microscopy has become an essential tool for studying the dynamical behaviors and variability of single cells, and cell-cell interactions. However, experiments and data analysis in this area are often extremely labor intensive, and it has often not been achievable or practical to perform properly standardized experiments on a statistically viable scale. We have addressed this challenge by developing automated live imaging platforms, to help standardize experiments, increasing throughput, and unlocking previously impossible ones. Our real-time cell tracking programs communicate in feedback with microscope and camera control software, and they are highly customizable, flexible, and efficient. As examples of our current research which utilize these automated platforms, we describe two quite different applications: egress-invasion interactions of malaria parasites and red blood cells, and imaging of immune cells which possess high motility and internal dynamics. The automated imaging platforms are able to track a large number of motile cells simultaneously, over hours or even days at a time, greatly increasing data throughput and opening up new experimental possibilities. Copyright © 2015 Elsevier Inc. All rights reserved.

GABAA receptor-mediated currents in interneurons and pyramidal cells of rat visual cortex

PubMed Central

Xiang, Zixiu; Huguenard, John R; Prince, David A

1998-01-01

We compared γ-aminobutyric acid (GABA)-mediated responses of identified pyramidal cells and fast spiking interneurons in layer V of visual cortical slices from young rats (P11-14). The frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) was similar in pyramidal cells and interneurons (1.7 vs. 1.9 Hz). For events with 10-90 % rise times less than 0.9 ms, no significant differences were found in mean amplitude (61 vs. 65 pA), mean rise time (0.58 vs. 0.61 ms), or the first time constant of decay (τ1, 6.4 vs. 6.5 ms) between pyramidal cells and interneurons. The second decay time constant (τ2) was significantly longer in interneurons than in pyramidal cells (49 vs. 22 ms). The difference in sIPSC decay kinetics between two cell types also existed in adult rats (P36-42), suggesting the kinetic difference is not due to differential development of GABAA receptors in these cell types. The decay kinetics of monosynaptic evoked IPSCs were also longer in interneurons. As in the case of sIPSCs, the difference was accounted for by the second decay time constant. τ1 and τ2 were, respectively, 13 and 64 ms for interneurons and 12 and 47 ms for pyramidal cells. Cell-attached patch recordings revealed that the mean open time for single Cl− channels in response to 2 μM GABA was significantly longer in interneurons than pyramidal cells (5.0 vs. 2.8 ms). The chord conductance of these channels in interneurons (12 pS) was significantly smaller than in pyramidal cells (15 pS). Single channel currents reversed polarity when the pipette potential was approximately -10 mV for both cell types. These results show that there is a functional diversity of GABAA receptors in electrophysiologically and morphologically identified cortical pyramidal cells and interneurons. This diversity might derive from the different molecular composition of the receptors in these two cell types. PMID:9503333

Circadian Clock Synchronization of the Cell Cycle in Zebrafish Occurs through a Gating Mechanism Rather Than a Period-phase Locking Process.

PubMed

Laranjeiro, Ricardo; Tamai, T Katherine; Letton, William; Hamilton, Noémie; Whitmore, David

2018-04-01

Studies from a number of model systems have shown that the circadian clock controls expression of key cell cycle checkpoints, thus providing permissive or inhibitory windows in which specific cell cycle events can occur. However, a major question remains: Is the clock actually regulating the cell cycle through such a gating mechanism or, alternatively, is there a coupling process that controls the speed of cell cycle progression? Using our light-responsive zebrafish cell lines, we address this issue directly by synchronizing the cell cycle in culture simply by changing the entraining light-dark (LD) cycle in the incubator without the need for pharmacological intervention. Our results show that the cell cycle rapidly reentrains to a shifted LD cycle within 36 h, with changes in p21 expression and subsequent S phase timing occurring within the first few hours of resetting. Reentrainment of mitosis appears to lag S phase resetting by 1 circadian cycle. The range of entrainment of the zebrafish clock to differing LD cycles is large, from 16 to 32 hour periods. We exploited this feature to explore cell cycle entrainment at both the population and single cell levels. At the population level, cell cycle length is shortened or lengthened under corresponding T-cycles, suggesting that a 1:1 coupling mechanism is capable of either speeding up or slowing down the cell cycle. However, analysis at the single cell level reveals that this, in fact, is not true and that a gating mechanism is the fundamental method of timed cell cycle regulation in zebrafish. Cell cycle length at the single cell level is virtually unaltered with varying T-cycles.

Circadian Clock Synchronization of the Cell Cycle in Zebrafish Occurs through a Gating Mechanism Rather Than a Period-phase Locking Process

PubMed Central

Tamai, T. Katherine; Letton, William; Hamilton, Noémie; Whitmore, David

2018-01-01

Studies from a number of model systems have shown that the circadian clock controls expression of key cell cycle checkpoints, thus providing permissive or inhibitory windows in which specific cell cycle events can occur. However, a major question remains: Is the clock actually regulating the cell cycle through such a gating mechanism or, alternatively, is there a coupling process that controls the speed of cell cycle progression? Using our light-responsive zebrafish cell lines, we address this issue directly by synchronizing the cell cycle in culture simply by changing the entraining light-dark (LD) cycle in the incubator without the need for pharmacological intervention. Our results show that the cell cycle rapidly reentrains to a shifted LD cycle within 36 h, with changes in p21 expression and subsequent S phase timing occurring within the first few hours of resetting. Reentrainment of mitosis appears to lag S phase resetting by 1 circadian cycle. The range of entrainment of the zebrafish clock to differing LD cycles is large, from 16 to 32 hour periods. We exploited this feature to explore cell cycle entrainment at both the population and single cell levels. At the population level, cell cycle length is shortened or lengthened under corresponding T-cycles, suggesting that a 1:1 coupling mechanism is capable of either speeding up or slowing down the cell cycle. However, analysis at the single cell level reveals that this, in fact, is not true and that a gating mechanism is the fundamental method of timed cell cycle regulation in zebrafish. Cell cycle length at the single cell level is virtually unaltered with varying T-cycles. PMID:29444612

High-content analysis of single cells directly assembled on CMOS sensor based on color imaging.

PubMed

Tanaka, Tsuyoshi; Saeki, Tatsuya; Sunaga, Yoshihiko; Matsunaga, Tadashi

2010-12-15

A complementary metal oxide semiconductor (CMOS) image sensor was applied to high-content analysis of single cells which were assembled closely or directly onto the CMOS sensor surface. The direct assembling of cell groups on CMOS sensor surface allows large-field (6.66 mm×5.32 mm in entire active area of CMOS sensor) imaging within a second. Trypan blue-stained and non-stained cells in the same field area on the CMOS sensor were successfully distinguished as white- and blue-colored images under white LED light irradiation. Furthermore, the chemiluminescent signals of each cell were successfully visualized as blue-colored images on CMOS sensor only when HeLa cells were placed directly on the micro-lens array of the CMOS sensor. Our proposed approach will be a promising technique for real-time and high-content analysis of single cells in a large-field area based on color imaging. Copyright © 2010 Elsevier B.V. All rights reserved.

In vivo marking of single cells in chick embryos using photoactivation of GFP.

PubMed

Stark, D A; Kulesa, P M

2005-10-01

Selective marking of a single cell within a living embryo is often difficult due to the inaccuracy and invasiveness of standard techniques. This unit describes a minimally invasive optical protocol that uses 405-nm laser light to photoactivate a variant of green fluorescent protein (PAGFP). This method takes advantage of the accessibility of the chick embryo to inject PAGFP into a region of interest and uses electroporation to deliver the construct into cells. This unit describes in detail how single and small groups of cells (n<10) that express PAGFP can be made visually distinguishable from the host population using the photoactivation process. Included is a means to maximize the fluorescence increase due to photoactivated GFP signal and to reduce photobleaching. Briefly outlined are previously developed chick culture and time-lapse imaging techniques to allow for the subsequent monitoring of photoactivated cell migratory behaviors. The technique has the potential to be a less-invasive, accurate tool for in vivo studies that involve following cell lineage and cell migration.

Order-of-magnitude estimates of latency (time to appearance) and refill time of a cancer from a single cancer 'stem' cell compared by an exponential and a logistic equation.

PubMed

Anderson, Ken M; Rubenstein, Marvin; Guinan, Patrick; Patel, Minu

2012-01-01

The time required before a mass of cancer cells considered to have originated from a single malignantly transformed cancer 'stem' cell reaches a certain number has not been studied. Applications might include determination of the time the cell mass reaches a size that can be detected by X-rays or physical examination or modeling growth rates in vitro in order to compare with other models or established data. We employed a simple logarithmic equation and a common logistic equation incorporating 'feedback' for unknown variables of cell birth, growth, division, and death that can be used to model cell proliferation. It can be used in association with free or commercial statistical software. Results with these two equations, varying the proliferation rate, nominally reduced by generational cell loss, are presented in two tables. The resulting equation, instructions, examples, and necessary mathematical software are available in the online appendix, where several parameters of interest can be modified by the reader www.uic.edu/nursing/publicationsupplements/tobillion_Anderson_Rubenstein_Guinan_Patel1.pdf. Reducing the proliferation rate by whatever alterations employed, markedly increases the time to reach 10(9) cells originating from an initial progenitor. In thinking about multistep oncogenesis, it is useful to consider the profound effect that variations in the effective proliferation rate may have during cancer development. This can be approached with the proposed equation, which is easy to use and available to further peer fine-tuning to be used in future modeling of cell growth.

Pathology Report for Intraperitoneal Sodium Dichromate Exposure in Rats, Protocol No. 15-002-3

DTIC Science & Technology

2015-12-08

neither was dose-dependent), subcapsular single cell necrosis was evident only at the highest dose, and minimal capsular fibroplasia in exposed...number per group of rats to exhibit any subcapsular granulocytic, mononuclear infiltrates, or single cell hepatocellular necrosis was greater, the higher...hepatocellular necrosis . Capsular fibrin appeared within 24 hours of injection but disappeared over time. Probably associated was the presence of

Properties of glutamate-gated ion channels in horizontal cells of the perch retina.

PubMed

Schmidt, K F

1997-08-01

The effect of two different concentrations of L-glutamate and kainate on the gating kinetics of amino acid-sensitive non-NMDA channels were studied in cultured teleost retinal horizontal cells by single-channel recording and by noise analysis of whole-cell currents. When the glutamate agonist kainate was applied clearly parabolic mean-variance relations of whole-cell membrane currents (up to 3000 pA) indicated that this agonist was acting on one type of channels with a conductance of 5-10 pS. The cells were less sensitive when L-glutamate was used as the agonist and in most cases whole-cell currents amounted to less than 200 pA. The mean-variance relation of glutamate induced currents was complex, indicating that more than one type of channel opening could be involved. Power spectra of whole-cell currents were fitted with two Lorentzians with time constants of approx. 1 and 5-20 msec. Effects on amplitudes and time constants of agonist concentrations are demonstrated. Two categories of unitary events with mean open times of approx. 1 and 7 msec and conductances of approx. 7 and 12 pS, respectively, were obtained in single-channel recordings from cell-attached patches at different concentrations of glutamate in the pipette.

Dual functional extracellular recording using a light-addressable potentiometric sensor for bitter signal transduction.

PubMed

Du, Liping; Wang, Jian; Chen, Wei; Zhao, Luhang; Wu, Chunsheng; Wang, Ping

2018-08-31

This paper presents a dual functional extracellular recording biosensor based on a light-addressable potentiometric sensor (LAPS). The design and fabrication of this biosensor make it possible to record both extracellular membrane potential changes and ATP release from a single taste bud cell for the first time. For detecting ATP release, LAPS chip was functionalized with ATP-sensitive DNA aptamer by covalent immobilization. Taste bud cells isolated from rat were cultured on LAPS surface. When the desired single taste bud cell was illuminated by modulated light, ATP release from single taste bud cells can be measured by recording the shifts of bias voltage-photocurrent curves (I-V curves) when the LAPS chip is working in discrete mode. On the other hand, extracellular membrane potential changes can be monitored by recording the fluctuation of LAPS photocurrent when the LAPS chip is working in continuous mode. The results show this biosensor can effectively record the enhancive effect of the bitter substance and inhibitory effect of the carbenoxolone (CBX) on the extracellular membrane potential changes and ATP release of single taste bud cells. In addition, the inhibitory effect of CBX also confirms LAPS extracellular recordings are originated from bitter signal transduction. It is proved this biosensor is suitable for extracellular recording of ATP release and membrane potential changes of single taste bud cells. It is suggested this biosensor could be applied to investigating taste signal transduction at the single-cell level as well as applied to other types of cells which have similar functions to taste bud cells. Copyright © 2018 Elsevier B.V. All rights reserved.

Hematopoietic stem cells derived from human umbilical cord ameliorate cisplatin-induced acute renal failure in rats

PubMed Central

Shalaby, Rokaya H; Rashed, Laila A; Ismaail, Alaa E; Madkour, Naglaa K; Elwakeel, Sherien H

2014-01-01

Injury to a target organ can be sensed by bone marrow stem cells that migrate to the site of damage, undergo differentiation, and promote structural and functional repair. This remarkable stem cell capacity prompted an investigation of the potential of mesenchymal and hematopoietic stem cells to cure acute renal failure. On the basis of the recent demonstration that hematopoietic stem cells (HSCs) can differentiate into renal cells, the current study tested the hypothesis that HSCs can contribute to the regeneration of renal tubular epithelial cells after renal injury. HSCs from human umbilical cord blood which isolated and purified by magnetic activated cell sorting were transplanted intraperitoneal into acute renal failure (ARF) rats which was established by a single dose of cisplatin 5 mg/kg for five days. The Study was carried on 48 male white albino rats, of average weight 120-150 gm. The animals were divided into 4 groups, Group one Served as control and received normal saline throughout the experiments. Group two (model control) received a single dose of cisplatin. Group three and four male-albino rats with induced ARF received interapritoneally (HSCs) at two week and four week respectively. Injection of a single dose of cisplatin resulted in a significant increase in serum creatinine and urea levels, histo-pathological examination of kidney tissue from cisplatin showed severe nephrotoxicity in which 50-75% of glomeruli and renal tubules exhibited massive degenerative change. Four weeks after HSC transplantation, Serum creatinine and urea nitrogen decreased 3.5 times and 2.1 times as well as HGF, IGF-1, VEGF and P53 using quantitative real-time PCR increased 4.3 times, 3.2, 2.4 and 4.2 times compared to ARF groups, respectively. The proliferation of cell nuclear antigen (PCNA)-positive cells (500.083±35.167) was higher than that in the cisplatin groups (58.612±15.743). In addition, the transplanted umbilical cord hematopoietic stem cells UC-HSCs could reside in local injury sites, leading to the relief of hyperemia and inflammation, but no obvious transdifferentiation into renal-like cells. The results lay the foundation for further study on the potential application of UC-HSCs in human disease and Because of their availability; HSC may be useful for cell replacement therapy of acute renal failure. PMID:25232508

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

PubMed Central

Rodriguez-Tirado, Carolina; Kitamura, Takanori; Kato, Yu; Pollard, Jeffery W.; Condeelis, John S.; Entenberg, David

2017-01-01

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell's arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature. PMID:27768066

A single-molecule view of gene regulation in cancer

NASA Astrophysics Data System (ADS)

Larson, Daniel

2013-03-01

Single-cell analysis has revealed that transcription is dynamic and stochastic, but tools are lacking that can determine the mechanism operating at a single gene. Here we utilize single-molecule observations of RNA in fixed and living cells to develop a single-cell model of steroid-receptor mediated gene activation. Steroid receptors coordinate a diverse range of responses in higher eukaryotes and are involved in a wide range of human diseases, including cancer. Steroid receptor response elements are present throughout the human genome and modulate chromatin remodeling and transcription in both a local and long-range fashion. As such, steroid receptor-mediated transcription is a paradigm of genetic control in the metazoan nucleus. Moreover, the ligand-dependent nature of these transcription factors makes them appealing targets for therapeutic intervention, necessitating a quantitative understanding of how receptors control output from target genes. We determine that steroids drive mRNA synthesis by frequency modulation of transcription. This digital behavior in single cells gives rise to the well-known analog dose response across the population. To test this model, we developed a light-activation technology to turn on a single gene and follow dynamic synthesis of RNA from the activated locus. The response delay is a measure of time required for chromatin remodeling at a single gene.

Single-cell real-time imaging of transgene expression upon lipofection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fiume, Giuseppe; Di Rienzo, Carmine; NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Piazza San Silvestro 12, 56127, Pisa

2016-05-20

Here we address the process of lipofection by quantifying the expression of a genetically-encoded fluorescent reporter at the single-cell level, and in real-time, by confocal imaging in live cells. The Lipofectamine gold-standard formulation is compared to the alternative promising DC-Chol/DOPE formulation. In both cases, we report that only dividing cells are able to produce a detectable amount of the fluorescent reporter protein. Notably, by measuring fluorescence over time in each pair of daughter cells, we find that Lipofectamine-based transfection statistically yields a remarkably higher degree of “symmetry” in protein expression between daughter cells as compared to DC-Chol/DOPE. A model ismore » envisioned in which the degree of symmetry of protein expression is linked to the number of bioavailable DNA copies within the cell before nuclear breakdown. Reported results open new perspectives for the understanding of the lipofection mechanism and define a new experimental platform for the quantitative comparison of transfection reagents. -- Highlights: •The process of lipofection is followed by quantifying the transgene expression in real time. •The Lipofectamine gold-standard is compared to the promising DC-Chol/DOPE formulation. •We report that only dividing cells are able to produce the fluorescent reporter protein. •The degree of symmetry of protein expression in daughter cells is linked to DNA bioavailability. •A new experimental platform for the quantitative comparison of transfection reagents is proposed.« less

Regulation of Hemopoietic Stem Cell Turnover and Population Size in Neonatal Mice

DTIC Science & Technology

1975-04-01

Following birth the hematopoietic stem cell population of the liver as measured by the in vivo spleen nodule assay (CFU) declines with a halving time...of about 48 hours. The stem cell population of the spleen grows exponentially with a doubling time of about 17 hours. In vitro incubation with high...single spleen colonies derived from neonatal liver and spleen CFU that both stem cell populations have a high self-renewal capacity. Thus, the decline in

Enhancing the reliability and throughput of neurosphere culture on hydrogel microwell arrays.

PubMed

Cordey, Myriam; Limacher, Monika; Kobel, Stefan; Taylor, Verdon; Lutolf, Matthias P

2008-10-01

The neurosphere assay is the standard retrospective assay to test the self-renewal capability and multipotency of neural stem cells (NSCs) in vitro. However, it has recently become clear that not all neurospheres are derived from a NSC and that on conventional cell culture substrates, neurosphere motility may cause frequent neurosphere "merging" [Nat Methods 2006;3:801-806; Stem Cells 2007;25:871-874]. Combining biomimetic hydrogel matrix technology with microengineering, we developed a microwell array platform on which NSC fate and neurosphere formation can be unequivocally attributed to a single founding cell. Using time-lapse microscopy and retrospective immunostaining, the fate of several hundred single NSCs was quantified. Compared with conventional neurosphere culture methods on plastic dishes, we detected a more than 100% increase in single NSC viability on soft hydrogels. Effective confinement of single proliferating cells to microwells led to neurosphere formation of vastly different sizes, a high percentage of which showed stem cell phenotypes after one week in culture. The reliability and increased throughput of this platform should help to better elucidate the function of sphere-forming stem/progenitor cells independent of their proliferation dynamics. Disclosure of potential conflicts of interest is found at the end of this article.

A novel anti-alpha-fetoprotein single-chain variable fragment displays anti-tumor effects in HepG2 cells as a single agent or in combination with paclitaxel.

PubMed

Ji, Xiaonan; Shen, Yanli; Sun, Hao; Gao, Xiangdong

2016-08-01

Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival time. The function of alpha-fetoprotein (AFP) as a regulatory factor in the growth of HCC cells has been well defined. The aim of this study was to investigate the use of a novel AFP-specific single-chain variable fragment that blocked AFP and inhibited HCC cell growth. The results indicated that the anti-AFP single-chain variable fragment (scFv) induced growth inhibition of AFP-expressing HCC cell lines in vitro through induction of G1 cell cycle arrest and apoptosis. The mechanism of apoptosis probably involved with blocking AFP internalization and regulation of the PTEN/PI3K/Akt signaling network. Moreover, the anti-AFP-scFv also effectively sensitized the HepG2 cells to paclitaxel (PTX) at a lower concentration. The combination effect of PTX and anti-AFP-scFv displayed a synergistic effect on HepG2 cells both in vitro and in vivo. Our results demonstrated that targeting AFP by specific antibodies has potential immunotherapeutic efficacy in human HCC.

High Resolution Measurement of the Glycolytic Rate

PubMed Central

Bittner, Carla X.; Loaiza, Anitsi; Ruminot, Iván; Larenas, Valeria; Sotelo-Hitschfeld, Tamara; Gutiérrez, Robin; Córdova, Alex; Valdebenito, Rocío; Frommer, Wolf B.; Barros, L. Felipe

2010-01-01

The glycolytic rate is sensitive to physiological activity, hormones, stress, aging, and malignant transformation. Standard techniques to measure the glycolytic rate are based on radioactive isotopes, are not able to resolve single cells and have poor temporal resolution, limitations that hamper the study of energy metabolism in the brain and other organs. A new method is described in this article, which makes use of a recently developed FRET glucose nanosensor to measure the rate of glycolysis in single cells with high temporal resolution. Used in cultured astrocytes, the method showed for the first time that glycolysis can be activated within seconds by a combination of glutamate and K+, supporting a role for astrocytes in neurometabolic and neurovascular coupling in the brain. It was also possible to make a direct comparison of metabolism in neurons and astrocytes lying in close proximity, paving the way to a high-resolution characterization of brain energy metabolism. Single-cell glycolytic rates were also measured in fibroblasts, adipocytes, myoblasts, and tumor cells, showing higher rates for undifferentiated cells and significant metabolic heterogeneity within cell types. This method should facilitate the investigation of tissue metabolism at the single-cell level and is readily adaptable for high-throughput analysis. PMID:20890447

Imaging trace element distributions in single organelles and subcellular features

NASA Astrophysics Data System (ADS)

Kashiv, Yoav; Austin, Jotham R.; Lai, Barry; Rose, Volker; Vogt, Stefan; El-Muayed, Malek

2016-02-01

The distributions of chemical elements within cells are of prime importance in a wide range of basic and applied biochemical research. An example is the role of the subcellular Zn distribution in Zn homeostasis in insulin producing pancreatic beta cells and the development of type 2 diabetes mellitus. We combined transmission electron microscopy with micro- and nano-synchrotron X-ray fluorescence to image unequivocally for the first time, to the best of our knowledge, the natural elemental distributions, including those of trace elements, in single organelles and other subcellular features. Detected elements include Cl, K, Ca, Co, Ni, Cu, Zn and Cd (which some cells were supplemented with). Cell samples were prepared by a technique that minimally affects the natural elemental concentrations and distributions, and without using fluorescent indicators. It could likely be applied to all cell types and provide new biochemical insights at the single organelle level not available from organelle population level studies.

Collective cell migration of thyroid carcinoma cells: a beneficial ability to override unfavourable substrates.

PubMed

Lobastova, Liudmila; Kraus, Dominik; Glassmann, Alexander; Khan, Dilaware; Steinhäuser, Christian; Wolff, Christina; Veit, Nadine; Winter, Jochen; Probstmeier, Rainer

2017-02-01

Tumor cell invasion and metastasis are life threatening events. Invasive tumor cells tend to migrate as collective sheets. In the present in vitro study we aimed to (i) assess whether collective tumor cells gain benefits in their migratory potential compared to single cells and (ii) to identify its putative underlying molecular mechanisms. The migratory potential of single and collective carcinoma cells was assessed using video time lapse microscopy and cell migration assays in the absence and presence of seven potential gap junction inhibitors or the Rac1 inhibitor Z62954982. The perturbation of gap junctions was assessed using a dye diffusion assay. In addition, LDH-based cytotoxicity and RT-PCR-based expression analyses were performed. Whereas single breast, cervix and thyroid carcinoma cells were virtually immobile on unfavourable plastic surfaces, we found that they gained pronounced migratory capacities as collectives under comparable conditions. Thyroid carcinoma cells, that were studied in more detail, were found to express specific subsets of connexins and to form active gap junctions as revealed by dye diffusion analysis. Although all potential gap junction blockers suppressed intercellular dye diffusion in at least one of the cell lines tested, only two of them were found to inhibit collective cell migration and none of them to inhibit single cell migration. In the presence of the Rac1 inhibitor Z62954982 collective migration, but not single cell migration, was found to be reduced up to 20 %. Our data indicate that collective migration enables tumor cells to cross otherwise unfavourable substrate areas. This capacity seems to be independent of intercellular communication via gap junctions, whereas Rac1-dependent intracellular signalling seems to be essential.

Protein profiling of single epidermal cell types from Arabidopsis thaliana using surface-enhanced laser desorption and ionization technology.

PubMed

Ebert, Berit; Melle, Christian; Lieckfeldt, Elke; Zöller, Daniela; von Eggeling, Ferdinand; Fisahn, Joachim

2008-08-25

Here, we describe a novel approach for investigating differential protein expression within three epidermal cell types. In particular, 3000 single pavement, basal, and trichome cells from leaves of Arabidopsis thaliana were harvested by glass micro-capillaries. Subsequently, these single cell samples were joined to form pools of 100 individual cells and analyzed using the ProteinChip technology; SELDI: surface-enhanced laser desorption and ionization. As a result, numerous protein signals that were differentially expressed in the three epidermal cell types could be detected. One of these proteins was characterized by tryptical digestion and subsequent identification via tandem quadrupole-time of flight (Q-TOF) mass spectrometry. Down regulation of this sequenced small subunit precursor of ribulose-1,5 bisphosphate carboxylase(C) oxygenase(O) (RuBisCo) in trichome and basal cells indicates the sink status of these cell types that are located on the surface of A. thaliana source leaves. Based on the obtained protein profiles, we suggest a close functional relationship between basal and trichome cells at the protein level.

Repeated sampling of genes from a single cell - implications for gravitropism research

NASA Astrophysics Data System (ADS)

Scherp, P.; Hasenstein, K. H.

The need for repeated but independent extractions of mRNA from single cells and plant tissues prompted the development of Solid Phase Gene Extraction (SPGE, patent pending). Oligo dT18 coated glass needles hybridize during a 2 to 3 min sampling time with the poly A+ mRNA. The needle is withdrawn and can be used directly for RT-PCR. Because of the small probe size, no cytoplasm is lost and repeated sampling of the same cell is possible. SPGE of Chara rhizoids and internodal cells showed fluctuations of type and quantity of mRNA in specific areas of the cytoplasm of rhizoids and time-dependent gene expression in internodal cells as a function of light/dark intervals. Despite extensive cytoplasmic streaming, mRNA-samples taken in the vicinity of the nucleus revealed a higher variability than the distal ends of the cell. In rhizoids, the mRNA/cDNA varied between the different zones of cytoplasm. In Arabidopsis, we isolated cDNA species from root tips, shoots and leaves and determined their sequences. Growth studies on SPGE-sampled individuals showed that after a short recovery period, all sampled plants resumed growth with normal growth rates and graviresponse. The data indicate that SPGE is a powerful method to study gene expression in single cells and in tissues of higher plants with high spatial and temporal resolution. Supported by NASA: NAG 2-1423

Single-cell computational analysis of light harvesting in a flat-panel photo-bioreactor.

PubMed

Loomba, Varun; Huber, Gregor; von Lieres, Eric

2018-01-01

Flat-panel photo-bioreactors (PBRs) are customarily applied for investigating growth of microalgae. Optimal design and operation of such reactors is still a challenge due to complex non-linear combinations of various impact factors, particularly hydrodynamics, light irradiation, and cell metabolism. A detailed analysis of single-cell light reception can lead to novel insights into the complex interactions of light exposure and algae movement in the reactor. The combined impacts of hydrodynamics and light irradiation on algae cultivation in a flat-panel PBR were studied by tracing the light exposure of individual cells over time. Hydrodynamics and turbulent mixing in this air-sparged bioreactor were simulated using the Eulerian approach for the liquid phase and a slip model for the gas phase velocity profiles. The liquid velocity was then used for tracing single cells and their light exposure, using light intensity profiles obtained from solving the radiative transfer equation at different wavelengths. The residence times of algae cells in defined dark and light zones of the PBR were statistically analyzed for different algal concentrations and sparging rates. The results indicate poor mixing caused by the reactor design which can be only partially improved by increased sparging rates. The results provide important information for optimizing algal biomass productivity by improving bioreactor design and operation and can further be utilized for an in-depth analysis of algal growth by using advanced models of cell metabolism.

Kinetics of HIV-1 Latency Reversal Quantified on the Single-Cell Level Using a Novel Flow-Based Technique.

PubMed

Martrus, G; Niehrs, A; Cornelis, R; Rechtien, A; García-Beltran, W; Lütgehetmann, M; Hoffmann, C; Altfeld, M

2016-10-15

HIV-1 establishes a pool of latently infected cells early following infection. New therapeutic approaches aiming at diminishing this persisting reservoir by reactivation of latently infected cells are currently being developed and tested. However, the reactivation kinetics of viral mRNA and viral protein production, and their respective consequences for phenotypical changes in infected cells that might enable immune recognition, remain poorly understood. We adapted a novel approach to assess the dynamics of HIV-1 mRNA and protein expression in latently and newly infected cells on the single-cell level by flow cytometry. This technique allowed the simultaneous detection of gagpol mRNA, intracellular p24 Gag protein, and cell surface markers. Following stimulation of latently HIV-1-infected J89 cells with human tumor necrosis factor alpha (hTNF-α)/romidepsin (RMD) or HIV-1 infection of primary CD4(+) T cells, four cell populations were detected according to their expression levels of viral mRNA and protein. gagpol mRNA in J89 cells was quantifiable for the first time 3 h after stimulation with hTNF-α and 12 h after stimulation with RMD, while p24 Gag protein was detected for the first time after 18 h poststimulation. HIV-1-infected primary CD4(+) T cells downregulated CD4, BST-2, and HLA class I expression at early stages of infection, proceeding Gag protein detection. In conclusion, here we describe a novel approach allowing quantification of the kinetics of HIV-1 mRNA and protein synthesis on the single-cell level and phenotypic characterization of HIV-1-infected cells at different stages of the viral life cycle. Early after infection, HIV-1 establishes a pool of latently infected cells, which hide from the immune system. Latency reversal and immune-mediated elimination of these latently infected cells are some of the goals of current HIV-1 cure approaches; however, little is known about the HIV-1 reactivation kinetics following stimulation with latency-reversing agents. Here we describe a novel approach allowing for the first time quantification of the kinetics of HIV-1 mRNA and protein synthesis after latency reactivation or de novo infection on the single-cell level using flow cytometry. This new technique furthermore enabled the phenotypic characterization of latently infected and de novo-infected cells dependent on the presence of viral RNA or protein. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Optoelectronic tweezers integrated with lensfree holographic microscopy for wide-field interactive cell and particle manipulation on a chip.

PubMed

Huang, Kuo-Wei; Su, Ting-Wei; Ozcan, Aydogan; Chiou, Pei-Yu

2013-06-21

We demonstrate an optoelectronic tweezer (OET) coupled to a lensfree holographic microscope for real-time interactive manipulation of cells and micro-particles over a large field-of-view (FOV). This integrated platform can record the holographic images of cells and particles over the entire active area of a CCD sensor array, perform digital image reconstruction to identify target cells, dynamically track the positions of cells and particles, and project light beams to trigger light-induced dielectrophoretic forces to pattern and sort cells on a chip. OET technology has been previously shown to be capable of performing parallel single cell manipulation over a large area. However, its throughput has been bottlenecked by the number of cells that can be imaged within the limited FOV of a conventional microscope objective lens. Integrating lensfree holographic imaging with OET solves this fundamental FOV barrier, while also creating a compact on-chip cell/particle manipulation platform. Using this unique platform, we have successfully demonstrated real-time interactive manipulation of thousands of single cells and micro-particles over an ultra-large area of e.g., 240 mm(2) (i.e. 17.96 mm × 13.52 mm).

Revealing Early Steps of α2β1 Integrin-mediated Adhesion to Collagen Type I by Using Single-Cell Force Spectroscopy

PubMed Central

Taubenberger, Anna; Cisneros, David A.; Friedrichs, Jens; Puech, Pierre-Henri; Muller, Daniel J.

2007-01-01

We have characterized early steps of α2β1 integrin-mediated cell adhesion to a collagen type I matrix by using single-cell force spectroscopy. In agreement with the role of α2β1 as a collagen type I receptor, α2β1-expressing Chinese hamster ovary (CHO)-A2 cells spread rapidly on the matrix, whereas α2β1-negative CHO wild-type cells adhered poorly. Probing CHO-A2 cell detachment forces over a contact time range of 600 s revealed a nonlinear adhesion response. During the first 60 s, cell adhesion increased slowly, and forces associated with the smallest rupture events were consistent with the breakage of individual integrin–collagen bonds. Above 60 s, a fraction of cells rapidly switched into an activated adhesion state marked by up to 10-fold increased detachment forces. Elevated overall cell adhesion coincided with a rise of the smallest rupture forces above the value required to break a single-integrin–collagen bond, suggesting a change from single to cooperative receptor binding. Transition into the activated adhesion mode and the increase of the smallest rupture forces were both blocked by inhibitors of actomyosin contractility. We therefore propose a two-step mechanism for the establishment of α2β1-mediated adhesion as weak initial, single-integrin–mediated binding events are superseded by strong adhesive interactions involving receptor cooperativity and actomyosin contractility. PMID:17314408

Live-cell imaging reveals the dynamics and function of single-telomere TERRA molecules in cancer cells.

PubMed

Avogaro, Laura; Querido, Emmanuelle; Dalachi, Myriam; Jantsch, Michael F; Chartrand, Pascal; Cusanelli, Emilio

2018-04-16

Telomeres cap the ends of eukaryotic chromosomes, protecting them from degradation and erroneous recombination events which may lead to genome instability. Telomeres are transcribed giving rise to telomeric repeat-containing RNAs, called TERRA. The TERRA long noncoding RNAs have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. While TERRA RNAs are predominantly nuclear and localize at telomeres, little is known about the dynamics and function of TERRA molecules expressed from individual telomeres. Herein, we developed an assay to image endogenous TERRA molecules expressed from a single telomere in living human cancer cells. We show that single-telomere TERRA can be detected as TERRA RNA single particles which freely diffuse within the nucleus. Furthermore, TERRA molecules aggregate forming TERRA clusters. Three-dimensional size distribution and single particle tracking analyses revealed distinct sizes and dynamics for TERRA RNA single particles and clusters. Simultaneous time lapse confocal imaging of TERRA particles and telomeres showed that TERRA clusters transiently co-localize with telomeres. Finally, we used chemically modified antisense oligonucleotides to deplete TERRA molecules expressed from a single telomere. Single-telomere TERRA depletion resulted in increased DNA damage at telomeres and elsewhere in the genome. These results suggest that single-telomere TERRA transcripts participate in the maintenance of genomic integrity in human cancer cells.

Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time.

PubMed

Wollman, Adam J M; Leake, Mark C

2015-01-01

We present a single-molecule tool called the CoPro (concentration of proteins) method that uses millisecond imaging with convolution analysis, automated image segmentation and super-resolution localization microscopy to generate robust estimates for protein concentration in different compartments of single living cells, validated using realistic simulations of complex multiple compartment cell types. We demonstrate its utility experimentally on model Escherichia coli bacteria and Saccharomyces cerevisiae budding yeast cells, and use it to address the biological question of how signals are transduced in cells. Cells in all domains of life dynamically sense their environment through signal transduction mechanisms, many involving gene regulation. The glucose sensing mechanism of S. cerevisiae is a model system for studying gene regulatory signal transduction. It uses the multi-copy expression inhibitor of the GAL gene family, Mig1, to repress unwanted genes in the presence of elevated extracellular glucose concentrations. We fluorescently labelled Mig1 molecules with green fluorescent protein (GFP) via chromosomal integration at physiological expression levels in living S. cerevisiae cells, in addition to the RNA polymerase protein Nrd1 with the fluorescent protein reporter mCherry. Using CoPro we make quantitative estimates of Mig1 and Nrd1 protein concentrations in the cytoplasm and nucleus compartments on a cell-by-cell basis under physiological conditions. These estimates indicate a ∼4-fold shift towards higher values in the concentration of diffusive Mig1 in the nucleus if the external glucose concentration is raised, whereas equivalent levels in the cytoplasm shift to smaller values with a relative change an order of magnitude smaller. This compares with Nrd1 which is not involved directly in glucose sensing, and which is almost exclusively localized in the nucleus under high and low external glucose levels. CoPro facilitates time-resolved quantification of protein concentrations in single functional cells, and enables the distributions of concentrations across a cell population to be measured. This could be useful in investigating several cellular processes that are mediated by proteins, especially where changes in protein concentration in a single cell in response to changes in the extracellular chemical environment are subtle and rapid and may be smaller than the variability across a cell population.

Real-time single cell analysis of molecular mechanism of apoptosis and proliferation using FRET technique

NASA Astrophysics Data System (ADS)

Chen, Tongsheng; Xing, Da; Gao, Xuejuan; Wang, Fang

2006-09-01

Bcl-2 family proteins (such as Bid and Bak/Bax) and 14-3-3 proteins play a key role in the mitochondria-mediated cell apoptosis induced by cell death factors such as TNF-α and lower power laser irradiation (LPLI). In this report, fluorescence resonance energy transfer (FRET) has been used to study the molecular mechanism of apoptosis in living cells on a fluorescence scanning confocal microscope. Based on the genetic code technique and the green fluorescent proteins (GFPs), single-cell dynamic analysis of caspase3 activation, caspase8 activation, and PKCs activation are performed during apoptosis induced by laser irradiation in real-time. To investigate the cellular effect and mechanism of laser irradiation, human lung adenocarcinoma cells (ASTC-a-1) transfected with plasmid SCAT3 (pSCAT3)/ CKAR FRET reporter, were irradiated and monitored noninvasively with both FRET imaging. Our results show that high fluence lower power laser irradiation (HFLPLI) can induce an increase of caspase3 activation and a decrease of PKCs activation, and that LPLI induces the ASTC-a-1 cell proliferation by specifically activating PKCs.

Real-time single cell analysis of Bid cleavage and translocation in cisplatin-induced apoptosis

NASA Astrophysics Data System (ADS)

Liu, Lei; Xing, Da; Pei, Yihui; Chen, Wei R.

2007-02-01

Cancer cell apoptosis can be induced by cisplatin, an efficient anticancer agent. However, its mechanism is not fully understood. Bcl-2 homology domain (BH) 3-only proteins couple stress signals to mitochondrial apoptotic pathways. Calpain-mediated cleavage of the BH3-only protein Bid into a 14 kD truncated protein (tBid) has been implicated in cisplatin-induced apoptotic pathway. We utilized a recombinant fluorescence resonance energy transfer (FRET) Bid probe to determine the kinetics of Bid cleavage during cisplatin-induced apoptosis in ASTC-a-1 cells. The cells were also co-transfected with Bid-CFP and DsRed-Mit to dynamically detect tBid translocation. Cells showed a cleavage of the Bid-FRET probe occurring at about 4-5 h after treated with 20 µM cisplatin. Cleavage of the Bid-FRET probe coincided with a translocation of tBid from the cytosolic to the mitochondria, and the translocation lasted about 1.5 h. Using real-time single-cell analysis, we first observed the kinetics of Bid cleavage and translocation to mitochondria in living cells during cisplatin-induced apoptosis.

Poisson-event-based analysis of cell proliferation.

PubMed

Summers, Huw D; Wills, John W; Brown, M Rowan; Rees, Paul

2015-05-01

A protocol for the assessment of cell proliferation dynamics is presented. This is based on the measurement of cell division events and their subsequent analysis using Poisson probability statistics. Detailed analysis of proliferation dynamics in heterogeneous populations requires single cell resolution within a time series analysis and so is technically demanding to implement. Here, we show that by focusing on the events during which cells undergo division rather than directly on the cells themselves a simplified image acquisition and analysis protocol can be followed, which maintains single cell resolution and reports on the key metrics of cell proliferation. The technique is demonstrated using a microscope with 1.3 μm spatial resolution to track mitotic events within A549 and BEAS-2B cell lines, over a period of up to 48 h. Automated image processing of the bright field images using standard algorithms within the ImageJ software toolkit yielded 87% accurate recording of the manually identified, temporal, and spatial positions of the mitotic event series. Analysis of the statistics of the interevent times (i.e., times between observed mitoses in a field of view) showed that cell division conformed to a nonhomogeneous Poisson process in which the rate of occurrence of mitotic events, λ exponentially increased over time and provided values of the mean inter mitotic time of 21.1 ± 1.2 hours for the A549 cells and 25.0 ± 1.1 h for the BEAS-2B cells. Comparison of the mitotic event series for the BEAS-2B cell line to that predicted by random Poisson statistics indicated that temporal synchronisation of the cell division process was occurring within 70% of the population and that this could be increased to 85% through serum starvation of the cell culture. © 2015 International Society for Advancement of Cytometry.

Light-sheet Bayesian microscopy enables deep-cell super-resolution imaging of heterochromatin in live human embryonic stem cells.

PubMed

Hu, Ying S; Zhu, Quan; Elkins, Keri; Tse, Kevin; Li, Yu; Fitzpatrick, James A J; Verma, Inder M; Cang, Hu

2013-01-01

Heterochromatin in the nucleus of human embryonic cells plays an important role in the epigenetic regulation of gene expression. The architecture of heterochromatin and its dynamic organization remain elusive because of the lack of fast and high-resolution deep-cell imaging tools. We enable this task by advancing instrumental and algorithmic implementation of the localization-based super-resolution technique. We present light-sheet Bayesian super-resolution microscopy (LSBM). We adapt light-sheet illumination for super-resolution imaging by using a novel prism-coupled condenser design to illuminate a thin slice of the nucleus with high signal-to-noise ratio. Coupled with a Bayesian algorithm that resolves overlapping fluorophores from high-density areas, we show, for the first time, nanoscopic features of the heterochromatin structure in both fixed and live human embryonic stem cells. The enhanced temporal resolution allows capturing the dynamic change of heterochromatin with a lateral resolution of 50-60 nm on a time scale of 2.3 s. Light-sheet Bayesian microscopy opens up broad new possibilities of probing nanometer-scale nuclear structures and real-time sub-cellular processes and other previously difficult-to-access intracellular regions of living cells at the single-molecule, and single cell level.

Light-sheet Bayesian microscopy enables deep-cell super-resolution imaging of heterochromatin in live human embryonic stem cells

PubMed Central

Hu, Ying S; Zhu, Quan; Elkins, Keri; Tse, Kevin; Li, Yu; Fitzpatrick, James A J; Verma, Inder M; Cang, Hu

2016-01-01

Background Heterochromatin in the nucleus of human embryonic cells plays an important role in the epigenetic regulation of gene expression. The architecture of heterochromatin and its dynamic organization remain elusive because of the lack of fast and high-resolution deep-cell imaging tools. We enable this task by advancing instrumental and algorithmic implementation of the localization-based super-resolution technique. Results We present light-sheet Bayesian super-resolution microscopy (LSBM). We adapt light-sheet illumination for super-resolution imaging by using a novel prism-coupled condenser design to illuminate a thin slice of the nucleus with high signal-to-noise ratio. Coupled with a Bayesian algorithm that resolves overlapping fluorophores from high-density areas, we show, for the first time, nanoscopic features of the heterochromatin structure in both fixed and live human embryonic stem cells. The enhanced temporal resolution allows capturing the dynamic change of heterochromatin with a lateral resolution of 50–60 nm on a time scale of 2.3 s. Conclusion Light-sheet Bayesian microscopy opens up broad new possibilities of probing nanometer-scale nuclear structures and real-time sub-cellular processes and other previously difficult-to-access intracellular regions of living cells at the single-molecule, and single cell level. PMID:27795878

In vitro analysis of low-level laser irradiation on human osteoblast-like cells proliferation

NASA Astrophysics Data System (ADS)

Bloise, Nora; Saino, Enrica; Bragheri, Francesca; Minzioni, Paolo; Cristiani, Ilaria; Imbriani, Marcello; Visai, Livia

2011-07-01

The objective of this study was to examine the in vitro effect of a single or a multiple doses of low-level laser irradiation (LLLI) on proliferation of the human osteosarcoma cell line, SAOS-2. SAOS-2 cells were divided in five groups and exposed to LLLI (659 nm diode laser; 11 mW power output): group I as a control (dark), group II exposed to a single laser dose of 1 J/cm2, group III irradiated with a single dose of 3 J/cm2, and group IV and V exposed for three consecutive days to 1 or 3 J/cm², respectively. Cellular proliferation was assessed daily up to 7 days of culturing. The obtained results showed an increase in proliferative capacity of SAOS-2 cells during the first 96 h of culturing time in once-irradiated cells, as compared to control cells. Furthermore, a significantly higher proliferation in the group IV and V was detected if compared to a single dose or to control group after 96 h and 7 days. In conclusion, the effect of the single dose on cell proliferation was transitory and repeated irradiations were necessary to observe a strong enhancement of SAOS-2 growth. As a future perspective, we would like to determine the potential of LLLI as a new approach for promoting bone regeneration onto biomaterials.

Mutagenic consequences of a single G-quadruplex demonstrate mitotic inheritance of DNA replication fork barriers

PubMed Central

Lemmens, Bennie; van Schendel, Robin; Tijsterman, Marcel

2015-01-01

Faithful DNA replication is vital to prevent disease-causing mutations, chromosomal aberrations and malignant transformation. However, accuracy conflicts with pace and flexibility and cells rely on specialized polymerases and helicases to ensure effective and timely replication of genomes that contain DNA lesions or secondary structures. If and how cells can tolerate a permanent barrier to replication is, however, unknown. Here we show that a single unresolved G-quadruplexed DNA structure can persist through multiple mitotic divisions without changing conformation. Failed replication across a G-quadruplex causes single-strand DNA gaps that give rise to DNA double-strand breaks in subsequent cell divisions, which are processed by polymerase theta (POLQ)-mediated alternative end joining. Lineage tracing experiments further reveal that persistent G-quadruplexes cause genetic heterogeneity during organ development. Our data demonstrate that a single lesion can cause multiple unique genomic rearrangements, and that alternative end joining enables cells to proliferate in the presence of mitotically inherited replication blocks. PMID:26563448

Mutagenic consequences of a single G-quadruplex demonstrate mitotic inheritance of DNA replication fork barriers.

PubMed

Lemmens, Bennie; van Schendel, Robin; Tijsterman, Marcel

2015-11-13

Faithful DNA replication is vital to prevent disease-causing mutations, chromosomal aberrations and malignant transformation. However, accuracy conflicts with pace and flexibility and cells rely on specialized polymerases and helicases to ensure effective and timely replication of genomes that contain DNA lesions or secondary structures. If and how cells can tolerate a permanent barrier to replication is, however, unknown. Here we show that a single unresolved G-quadruplexed DNA structure can persist through multiple mitotic divisions without changing conformation. Failed replication across a G-quadruplex causes single-strand DNA gaps that give rise to DNA double-strand breaks in subsequent cell divisions, which are processed by polymerase theta (POLQ)-mediated alternative end joining. Lineage tracing experiments further reveal that persistent G-quadruplexes cause genetic heterogeneity during organ development. Our data demonstrate that a single lesion can cause multiple unique genomic rearrangements, and that alternative end joining enables cells to proliferate in the presence of mitotically inherited replication blocks.

Shrink-induced single-cell plastic microwell array.

PubMed

Lew, Valerie; Nguyen, Diep; Khine, Michelle

2011-12-01

The ability to interrogate and track single cells over time in a high-throughput format would provide critical information for fundamental biological understanding of processes and for various applications, including drug screening and toxicology. We have developed an ultrarapid and simple method to create single-cell wells of controllable diameter and depth with commodity shrink-wrap film and tape. Using a programmable CO(2) laser, we cut hole arrays into the tape. The tape then serves as a shadow mask to selectively etch wells into commodity shrink-wrap film by O(2) plasma. When the shrink-wrap film retracts upon briefly heating, high-aspect plastic microwell arrays with diameters down to 20 μm are readily achieved. We calibrated the loading procedure with fluorescent microbeads. Finally, we demonstrate the utility of the wells by loading fluorescently labeled single human embryonic stem cells into the wells. Copyright © 2011 Society for Laboratory Automation and Screening. Published by Elsevier Inc. All rights reserved.

Single-cell topological RNA-Seq analysis reveals insights into cellular differentiation and development

PubMed Central

Rizvi, Abbas H.; Camara, Pablo G.; Kandror, Elena K.; Roberts, Thomas J.; Schieren, Ira; Maniatis, Tom; Rabadan, Raul

2017-01-01

Transcriptional programs control cellular lineage commitment and differentiation during development. Understanding cell fate has been advanced by studying single-cell RNA-seq, but is limited by the assumptions of current analytic methods regarding the structure of data. We present single-cell topological data analysis (scTDA), an algorithm for topology-based computational analyses to study temporal, unbiased transcriptional regulation. Compared to other methods, scTDA is a non-linear, model-independent, unsupervised statistical framework that can characterize transient cellular states. We applied scTDA to the analysis of murine embryonic stem cell (mESC) differentiation in vitro in response to inducers of motor neuron differentiation. scTDA resolved asynchrony and continuity in cellular identity over time, and identified four transient states (pluripotent, precursor, progenitor, and fully differentiated cells) based on changes in stage-dependent combinations of transcription factors, RNA-binding proteins and long non-coding RNAs. scTDA can be applied to study asynchronous cellular responses to either developmental cues or environmental perturbations. PMID:28459448

Granatum: a graphical single-cell RNA-Seq analysis pipeline for genomics scientists.

PubMed

Zhu, Xun; Wolfgruber, Thomas K; Tasato, Austin; Arisdakessian, Cédric; Garmire, David G; Garmire, Lana X

2017-12-05

Single-cell RNA sequencing (scRNA-Seq) is an increasingly popular platform to study heterogeneity at the single-cell level. Computational methods to process scRNA-Seq data are not very accessible to bench scientists as they require a significant amount of bioinformatic skills. We have developed Granatum, a web-based scRNA-Seq analysis pipeline to make analysis more broadly accessible to researchers. Without a single line of programming code, users can click through the pipeline, setting parameters and visualizing results via the interactive graphical interface. Granatum conveniently walks users through various steps of scRNA-Seq analysis. It has a comprehensive list of modules, including plate merging and batch-effect removal, outlier-sample removal, gene-expression normalization, imputation, gene filtering, cell clustering, differential gene expression analysis, pathway/ontology enrichment analysis, protein network interaction visualization, and pseudo-time cell series construction. Granatum enables broad adoption of scRNA-Seq technology by empowering bench scientists with an easy-to-use graphical interface for scRNA-Seq data analysis. The package is freely available for research use at http://garmiregroup.org/granatum/app.

Live cell isolation by laser microdissection with gravity transfer

NASA Astrophysics Data System (ADS)

Podgorny, Oleg V.

2013-05-01

Laser microdissection by pulsing ultraviolet laser allows the isolation and recultivation of live cells based on morphological features or/and fluorescent labelling from adherent cell cultures. Previous investigations described only the use of the laser microdissection and pressure catapulting (LMPC) for live cell isolation. But LMPC requires complex manipulations and some skill. Furthermore, single-cell cloning using laser microdissection has not yet been demonstrated. The first evidence of successful application of laser microdissection with gravity transfer (LMDGT) for capturing and recultivation of live cells is presented. A new strategy for LMDGT is presented because of the failure to reproduce the manufacturer's protocol. Using the new strategy, successful capturing and recultivation of circle-shaped samples from confluent monolayer of HeLa cells was demonstrated. It was found that LMDGT is easier than LMPC because it doesn't require personal participation of investigator in transferring of isolated samples to final culture dishes. Moreover, for the first time, the generation of clonal colonies from single live cells isolated by laser microdissection was demonstrated. Data obtained in this study confirm that LMDGT is a reliable and high-yield method allowing isolation and expansion of both cell clusters and single cells from adherent cell cultures.

Adhesion and migration of CHO cells on micropatterned single layer graphene

NASA Astrophysics Data System (ADS)

Keshavan, S.; Oropesa-Nuñez, R.; Diaspro, A.; Canale, C.; Dante, S.

2017-06-01

Cell patterning technology on single layer graphene (SLG) is a fairly new field that can find applications in tissue engineering and biomaterial/biosensors development. Recently, we have developed a simple and effective approach for the fabrication of patterned SLG substrates by laser micromachining, and we have successfully applied it for the obtainment of geometrically ordered neural networks. Here, we exploit the same approach to investigate the generalization of the cell response to the surface cues of the fabricated substrates and, contextually, to quantify cell adhesion on the different areas of the patterns. To attain this goal, we tested Chinese hamster ovary (CHO) cells on PDL-coated micropatterned SLG substrates and quantified the adhesion by using single cell force spectroscopy (SCFS). Our results indicate higher cell adhesion on PDL-SLG, and, consequently, an initial CHO cell accumulation on the graphene areas, confirming the neuronal behaviour observed previously; interestingly, at later time point in culture, cell migration was observed towards the adjacent SLG ablated regions, which resulted more favourable for cell proliferation. Therefore, our findings indicate that the mechanism of interaction with the surface cues offered by the micropatterned substrates is strictly cell-type dependent.

Emergent Phototactic Responses of Cyanobacteria under Complex Light Regimes

PubMed Central

Chau, Rosanna Man Wah

2017-01-01

ABSTRACT Environmental cues can stimulate a variety of single-cell responses, as well as collective behaviors that emerge within a bacterial community. These responses require signal integration and transduction, which can occur on a variety of time scales and often involve feedback between processes, for example, between growth and motility. Here, we investigate the dynamics of responses of the phototactic, unicellular cyanobacterium Synechocystis sp. PCC6803 to complex light inputs that simulate the natural environments that cells typically encounter. We quantified single-cell motility characteristics in response to light of different wavelengths and intensities. We found that red and green light primarily affected motility bias rather than speed, while blue light inhibited motility altogether. When light signals were simultaneously presented from different directions, cells exhibited phototaxis along the vector sum of the light directions, indicating that cells can sense and combine multiple signals into an integrated motility response. Under a combination of antagonistic light signal regimes (phototaxis-promoting green light and phototaxis-inhibiting blue light), the ensuing bias was continuously tuned by competition between the wavelengths, and the community response was dependent on both bias and cell growth. The phototactic dynamics upon a rapid light shift revealed a wavelength dependence on the time scales of photoreceptor activation/deactivation. Thus, Synechocystis cells achieve exquisite integration of light inputs at the cellular scale through continuous tuning of motility, and the pattern of collective behavior depends on single-cell motility and population growth. PMID:28270586

Quantitative imaging of mammalian transcriptional dynamics: from single cells to whole embryos.

PubMed

Zhao, Ziqing W; White, Melanie D; Bissiere, Stephanie; Levi, Valeria; Plachta, Nicolas

2016-12-23

Probing dynamic processes occurring within the cell nucleus at the quantitative level has long been a challenge in mammalian biology. Advances in bio-imaging techniques over the past decade have enabled us to directly visualize nuclear processes in situ with unprecedented spatial and temporal resolution and single-molecule sensitivity. Here, using transcription as our primary focus, we survey recent imaging studies that specifically emphasize the quantitative understanding of nuclear dynamics in both time and space. These analyses not only inform on previously hidden physical parameters and mechanistic details, but also reveal a hierarchical organizational landscape for coordinating a wide range of transcriptional processes shared by mammalian systems of varying complexity, from single cells to whole embryos.

Improvement of minority carrier life time in N-type monocrystalline Si by the Czochralski method

NASA Astrophysics Data System (ADS)

Baik, Sungsun; Pang, Ilsun; Kim, Jaemin; Kim, Kwanghun

2016-07-01

The installation amount of solar power plants increases every year. Multi-crystalline Si solar cells comprise a large share of the market of solar power plants. Multi-crystalline and single-crystalline Si solar cells are competing against one another in the market. Many single-crystalline companies are trying to develop and produce n-type solar cells with higher cell efficiency than that of p-type. In n-type wafers with high cell efficiency, wafer quality has become increasingly important. In order to make ingots with higher MCLT, the effects of both poly types related to metal impurities and pull speeds related to vacancy concentration on minority carrier life time were studied. In the final part of ingots, poly types related to the metal impurities are a dominant factor on MCLT. In the initial part of ingots, pull speeds related to vacancy concentration are a dominant factor on MCLT. [Figure not available: see fulltext.

Lights, Camera, Action! Antimicrobial Peptide Mechanisms Imaged in Space and Time

PubMed Central

Choi, Heejun; Rangarajan, Nambirajan; Weisshaar, James C.

2015-01-01

Deeper understanding of the bacteriostatic and bactericidal mechanisms of antimicrobial peptides (AMPs) should help in the design of new antibacterial agents. Over several decades, a variety of biochemical assays have been applied to bulk bacterial cultures. While some of these bulk assays provide time resolution on the order of 1 min, they do not capture faster mechanistic events. Nor can they provide subcellular spatial information or discern cell-to-cell heterogeneity within the bacterial population. Single-cell, time-resolved imaging assays bring a completely new spatiotemporal dimension to AMP mechanistic studies. We review recent work that provides new insights into the timing, sequence, and spatial distribution of AMP-induced effects on bacterial cells. PMID:26691950

Integrating discrete stochastic models and single-cell experiments to infer predictive models of MAPK-induced transcription dynamics

NASA Astrophysics Data System (ADS)

Munsky, Brian

2015-03-01

MAPK signal-activated transcription plays central roles in myriad biological processes including stress adaptation responses and cell fate decisions. Recent single-cell and single-molecule experiments have advanced our ability to quantify the spatial, temporal, and stochastic fluctuations for such signals and their downstream effects on transcription regulation. This talk explores how integrating such experiments with discrete stochastic computational analyses can yield quantitative and predictive understanding of transcription regulation in both space and time. We use single-molecule mRNA fluorescence in situ hybridization (smFISH) experiments to reveal locations and numbers of multiple endogenous mRNA species in 100,000's of individual cells, at different times and under different genetic and environmental perturbations. We use finite state projection methods to precisely and efficiently compute the full joint probability distributions of these mRNA, which capture measured spatial, temporal and correlative fluctuations. By combining these experimental and computational tools with uncertainty quantification, we systematically compare models of varying complexity and select those which give optimally precise and accurate predictions in new situations. We use these tools to explore two MAPK-activated gene regulation pathways. In yeast adaptation to osmotic shock, we analyze Hog1 kinase activation of transcription for three different genes STL1 (osmotic stress), CTT1 (oxidative stress) and HSP12 (heat shock). In human osteosarcoma cells under serum induction, we analyze ERK activation of c-Fos transcription.

Block-Cell-Printing for live single-cell printing

PubMed Central

Zhang, Kai; Chou, Chao-Kai; Xia, Xiaofeng; Hung, Mien-Chie; Qin, Lidong

2014-01-01

A unique live-cell printing technique, termed “Block-Cell-Printing” (BloC-Printing), allows for convenient, precise, multiplexed, and high-throughput printing of functional single-cell arrays. Adapted from woodblock printing techniques, the approach employs microfluidic arrays of hook-shaped traps to hold cells at designated positions and directly transfer the anchored cells onto various substrates. BloC-Printing has a minimum turnaround time of 0.5 h, a maximum resolution of 5 µm, close to 100% cell viability, the ability to handle multiple cell types, and efficiently construct protrusion-connected single-cell arrays. The approach enables the large-scale formation of heterotypic cell pairs with controlled morphology and allows for material transport through gap junction intercellular communication. When six types of breast cancer cells are allowed to extend membrane protrusions in the BloC-Printing device for 3 h, multiple biophysical characteristics of cells—including the protrusion percentage, extension rate, and cell length—are easily quantified and found to correlate well with their migration levels. In light of this discovery, BloC-Printing may serve as a rapid and high-throughput cell protrusion characterization tool to measure the invasion and migration capability of cancer cells. Furthermore, primary neurons are also compatible with BloC-Printing. PMID:24516129

A polymeric micro total analysis system for single-cell analysis

NASA Astrophysics Data System (ADS)

Lai, Hsuan-Hong

The advancement of microengineering has enabled the manipulation and analysis of single cells, which is critical in understanding the molecular mechanisms underlying the basic physiological functions from the point of view of modern biologists. Unfortunately, analysis of single cells remains challenging from a technical perspective, mainly because of the miniature nature of the cell and the high throughput requirements of the analysis. Lab-on-a-chip (LOC) emerges as a research field that shows great promise in this perspective. We have demonstrated a micro total analysis system (mu-TAS) combining chip-based electrophoretic separation, fluorescence detection, and a pulsed Nd:YAG laser cell lysis system, in a Poly(dimethylsiloxane) (PDMS) microfluidic analytical platform for the implementation of single-cell analysis. To accomplish the task, a polymeric microfluidic device was fabricated and UV graft polymerization surface modification techniques were used. To optimize the conditions for the surface treatment techniques, the modified surfaces of PDMS were characterized using AIR-IR spectrum and sessile water drop contact angle measurements, and in-channel surfaces were characterized by their electroosmotic flow mobility. Accurate single-cell analysis relies on rapid cell lysis and therefore an optical measure of fast cell lysis was implemented and optimized in a microscopic station. The influences of pulse energy and the location of the laser beam with respect to the cell in the microchannel were explored. The observation from the cell disruption experiments suggested that the cell lysis was enabled mainly via a thermo-mechanical instead of a plasma-mediated mechanism. Finally, after chip-based electrophoresis and a laser-induced fluorescence (LIF) detection system were incorporated with the laser lysis system in a microfluidic analytical station, a feasibility demonstration of single-cell analysis was implemented. The analytical platform exhibited the capability of fluidic transportation, optical lysis of single cells, separation, and analysis of the lysates by electrophoresis and LIF detection. In comparison with the control experiment, the migration times of the fluorescent signals for the cytosolic fluorophores were in good agreement with those for the standard fluorophores, which confirmed the feasibility of the analytical processes.

Electrochemical Quantification of Extracellular Local H2O2 Kinetics Originating from Single Cells.

PubMed

Bozem, Monika; Knapp, Phillip; Mirčeski, Valentin; Slowik, Ewa J; Bogeski, Ivan; Kappl, Reinhard; Heinemann, Christian; Hoth, Markus

2017-05-15

H 2 O 2 is produced by all eukaryotic cells under physiological and pathological conditions. Due to its enormous relevance for cell signaling at low concentrations and antipathogenic function at high concentrations, precise quantification of extracellular local H 2 O 2 concentrations ([H 2 O 2 ]) originating from single cells is required. Using a scanning electrochemical microscope and bare platinum disk ultramicroelectrodes, we established sensitive long-term measurements of extracellular [H 2 O 2 ] kinetics originating from single primary human monocytes (MCs) ex vivo. For the electrochemical techniques square wave voltammetry, cyclic and linear scan voltammetry, and chronoamperometry, detection limits for [H 2 O 2 ] were determined to be 5, 50, and 500 nM, respectively. Following phorbol ester stimulation, local [H 2 O 2 ] 5-8 μm above a single MC increased by 3.4 nM/s within the first 10 min before reaching a plateau. After extracellular addition of H 2 O 2 to an unstimulated MC, the local [H 2 O 2 ] decreased on average by 4.2 nM/s due to degradation processes of the cell. Using the scanning mode of the setup, we found that H 2 O 2 is evenly distributed around the producing cell and can still be detected up to 30 μm away from the cell. The electrochemical single-cell measurements were validated in MC populations using electron spin resonance spectroscopy and the Amplex ® UltraRed assay. Innovation and Conclusion: We demonstrate a highly sensitive, spatially, and temporally resolved electrochemical approach to monitor dynamics of production and degradation processes for H 2 O 2 separately. Local extracellular [H 2 O 2 ] kinetics originating from single cells is quantified in real time. Antioxid. Redox Signal. 00, 000-000.

Sensitive Optical and Microfluidic Systems for Cellular Analyses

NASA Astrophysics Data System (ADS)

Schiro, Perry G.

Investigating rare cells and heterogeneous subpopulations is challenging for a myriad reasons. This dissertation describes novel techniques to analyze single molecules, synaptic vesicles, and rare circulating tumor cells. The eDAR platform for isolating rare cells in fluids provides a new method to monitor breast cancer status in patients as well as to guide research for personalized treatment and efficacy. In a side-by-side comparison with CellSearch, eDAR detected CTCs in all 20 Stage IV metastatic breast cancer patients while the CellSearch system found CTCs in just 8 patients. The single-molecule capillary electrophoresis technology is a method to characterize an entire sample one molecule at a time, providing detailed information about the absolute number and nature of molecules present in a sample. The nFASS platform has the potential to apply the advantages that currently exist in flow cytometry to the study of items on a much smaller scale such as subcellular organelles and nanometer-sized objects. For example, the isolation of subpopulations of synaptic vesicles will allow for detailed protein quantification and identification in the study of neurological diseases. These tools facilitate fundamental investigation of objects ranging from single molecules to single cells.

Single dose of filgrastim (rhG-CSF) increases the number of hematopoietic progenitors in the peripheral blood of adult volunteers.

PubMed

Schwinger, W; Mache, C; Urban, C; Beaufort, F; Töglhofer, W

1993-06-01

Hematopoietic progenitor cell levels were monitored in the peripheral blood of ten healthy adults receiving a single dose of recombinant human granulocyte colony-stimulating factor (rhG-CSF). The objective was to determine the time and number of progenitor cells released into the peripheral blood, induced by a single dose of 15 micrograms/kg rhG-CSF administered intravenously. In all cases the absolute number of circulating progenitor cells including granulocyte-macrophage and erythroid lineages increased up to 12-fold (median 9.4-fold) 4 days after treatment. These findings were based on flow cytometric quantification of CD34+ cells and on progenitor assays. The relative distribution of granulocyte/macrophage and erythroid progenitors remained unchanged. rhG-CSF was well tolerated; mild to moderate bone pain was the most common side-effect and was noted in 6 of 10 subjects. Thus a single dose of rhG-CSF is effective in mobilizing progenitor cells into the peripheral blood in healthy adults. If these progenitors are capable of reconstituting bone marrow, peripheral progenitor cell separation following rhG-CSF administration could be a reasonable alternative to conventional bone marrow harvest in healthy adults.

A Multiplexed Assay That Monitors Effects of Multiple Compound Treatment Times Reveals Candidate Immune-Enhancing Compounds.

PubMed

Zhao, Ziyan; Henowitz, Liza; Zweifach, Adam

2018-05-01

We previously developed a flow cytometry assay that monitored lytic granule exocytosis in cytotoxic T lymphocytes stimulated by contacting beads coated with activating anti-CD3 antibodies. That assay was multiplexed in that responses of cells that did or did not receive the activating stimulus were distinguished via changes in light scatter accompanying binding of cells to beads, allowing us to discriminate compounds that activate responses on their own from compounds that enhance responses in cells that received the activating stimulus, all within a single sample. Here we add a second dimension of multiplexing by developing means to assess in a single sample the effects of treating cells with test compounds for different times. Bar-coding cells before adding them to test wells lets us determine compound treatment time while also monitoring activation status and response amplitude at the point of interrogation. This multiplexed assay is suitable for screening 96-well plates. We used it to screen compounds from the National Cancer Institute, identifying several compounds that enhance anti-LAMP1 responses. Multiple-treatment-time (MTT) screening enabled by bar-coding and read via high-throughput flow cytometry may be a generally useful method for facilitating the discovery of compounds of interest.

Meaningful interpretation of subdiffusive measurements in living cells (crowded environment) by fluorescence fluctuation microscopy.

PubMed

Baumann, Gerd; Place, Robert F; Földes-Papp, Zeno

2010-08-01

In living cell or its nucleus, the motions of molecules are complicated due to the large crowding and expected heterogeneity of the intracellular environment. Randomness in cellular systems can be either spatial (anomalous) or temporal (heterogeneous). In order to separate both processes, we introduce anomalous random walks on fractals that represented crowded environments. We report the use of numerical simulation and experimental data of single-molecule detection by fluorescence fluctuation microscopy for detecting resolution limits of different mobile fractions in crowded environment of living cells. We simulate the time scale behavior of diffusion times tau(D)(tau) for one component, e.g. the fast mobile fraction, and a second component, e.g. the slow mobile fraction. The less the anomalous exponent alpha the higher the geometric crowding of the underlying structure of motion that is quantified by the ratio of the Hausdorff dimension and the walk exponent d(f)/d(w) and specific for the type of crowding generator used. The simulated diffusion time decreases for smaller values of alpha # 1 but increases for a larger time scale tau at a given value of alpha # 1. The effect of translational anomalous motion is substantially greater if alpha differs much from 1. An alpha value close to 1 contributes little to the time dependence of subdiffusive motions. Thus, quantitative determination of molecular weights from measured diffusion times and apparent diffusion coefficients, respectively, in temporal auto- and crosscorrelation analyses and from time-dependent fluorescence imaging data are difficult to interpret and biased in crowded environments of living cells and their cellular compartments; anomalous dynamics on different time scales tau must be coupled with the quantitative analysis of how experimental parameters change with predictions from simulated subdiffusive dynamics of molecular motions and mechanistic models. We first demonstrate that the crowding exponent alpha also determines the resolution of differences in diffusion times between two components in addition to photophysical parameters well-known for normal motion in dilute solution. The resolution limit between two different kinds of single molecule species is also analyzed under translational anomalous motion with broken ergodicity. We apply our theoretical predictions of diffusion times and lower limits for the time resolution of two components to fluorescence images in human prostate cancer cells transfected with GFP-Ago2 and GFP-Ago1. In order to mimic heterogeneous behavior in crowded environments of living cells, we need to introduce so-called continuous time random walks (CTRW). CTRWs were originally performed on regular lattice. This purely stochastic molecule behavior leads to subdiffusive motion with broken ergodicity in our simulations. For the first time, we are able to quantitatively differentiate between anomalous motion without broken ergodicity and anomalous motion with broken ergodicity in time-dependent fluorescence microscopy data sets of living cells. Since the experimental conditions to measure a selfsame molecule over an extended period of time, at which biology is taken place, in living cells or even in dilute solution are very restrictive, we need to perform the time average over a subpopulation of different single molecules of the same kind. For time averages over subpopulations of single molecules, the temporal auto- and crosscorrelation functions are first found. Knowing the crowding parameter alpha for the cell type and cellular compartment type, respectively, the heterogeneous parameter gamma can be obtained from the measurements in the presence of the interacting reaction partner, e.g. ligand, with the same alpha value. The product alpha x gamma = gamma is not a simple fitting parameter in the temporal auto- and two-color crosscorrelation functions because it is related to the proper physical models of anomalous (spatial) and heterogeneous (temporal) randomness in cellular systems.We have already derived an analytical solution gamma for in the special case of gamma = 3/2. In the case of two-color crosscorrelation or/and two-color fluorescence imaging (co-localization experiments), the second component is also a two-color species gr, for example a different molecular complex with an additional ligand. Here, we first show that plausible biological mechanisms from FCS/ FCCS and fluorescence imaging in living cells are highly questionable without proper quantitative physical models of subdiffusive motion and temporal randomness. At best, such quantitative FCS/ FCCS and fluorescence imaging data are difficult to interpret under crowding and heterogeneous conditions. It is challenging to translate proper physical models of anomalous (spatial) and heterogeneous (temporal) randomness in living cells and their cellular compartments like the nucleus into biological models of the cell biological process under study testable by single-molecule approaches. Otherwise, quantitative FCS/FCCS and fluorescence imaging measurements in living cells are not well described and cannot be interpreted in a meaningful way.

A machine learning approach for the identification of key markers involved in brain development from single-cell transcriptomic data.

PubMed

Hu, Yongli; Hase, Takeshi; Li, Hui Peng; Prabhakar, Shyam; Kitano, Hiroaki; Ng, See Kiong; Ghosh, Samik; Wee, Lawrence Jin Kiat

2016-12-22

The ability to sequence the transcriptomes of single cells using single-cell RNA-seq sequencing technologies presents a shift in the scientific paradigm where scientists, now, are able to concurrently investigate the complex biology of a heterogeneous population of cells, one at a time. However, till date, there has not been a suitable computational methodology for the analysis of such intricate deluge of data, in particular techniques which will aid the identification of the unique transcriptomic profiles difference between the different cellular subtypes. In this paper, we describe the novel methodology for the analysis of single-cell RNA-seq data, obtained from neocortical cells and neural progenitor cells, using machine learning algorithms (Support Vector machine (SVM) and Random Forest (RF)). Thirty-eight key transcripts were identified, using the SVM-based recursive feature elimination (SVM-RFE) method of feature selection, to best differentiate developing neocortical cells from neural progenitor cells in the SVM and RF classifiers built. Also, these genes possessed a higher discriminative power (enhanced prediction accuracy) as compared commonly used statistical techniques or geneset-based approaches. Further downstream network reconstruction analysis was carried out to unravel hidden general regulatory networks where novel interactions could be further validated in web-lab experimentation and be useful candidates to be targeted for the treatment of neuronal developmental diseases. This novel approach reported for is able to identify transcripts, with reported neuronal involvement, which optimally differentiate neocortical cells and neural progenitor cells. It is believed to be extensible and applicable to other single-cell RNA-seq expression profiles like that of the study of the cancer progression and treatment within a highly heterogeneous tumour.

Pinched-flow hydrodynamic stretching of single-cells.

PubMed

Dudani, Jaideep S; Gossett, Daniel R; Tse, Henry T K; Di Carlo, Dino

2013-09-21

Reorganization of cytoskeletal networks, condensation and decondensation of chromatin, and other whole cell structural changes often accompany changes in cell state and can reflect underlying disease processes. As such, the observable mechanical properties, or mechanophenotype, which is closely linked to intracellular architecture, can be a useful label-free biomarker of disease. In order to make use of this biomarker, a tool to measure cell mechanical properties should accurately characterize clinical specimens that consist of heterogeneous cell populations or contain small diseased subpopulations. Because of the heterogeneity and potential for rare populations in clinical samples, single-cell, high-throughput assays are ideally suited. Hydrodynamic stretching has recently emerged as a powerful method for carrying out mechanical phenotyping. Importantly, this method operates independently of molecular probes, reducing cost and sample preparation time, and yields information-rich signatures of cell populations through significant image analysis automation, promoting more widespread adoption. In this work, we present an alternative mode of hydrodynamic stretching where inertially-focused cells are squeezed in flow by perpendicular high-speed pinch flows that are extracted from the single inputted cell suspension. The pinched-flow stretching method reveals expected differences in cell deformability in two model systems. Furthermore, hydraulic circuit design is used to tune stretching forces and carry out multiple stretching modes (pinched-flow and extensional) in the same microfluidic channel with a single fluid input. The ability to create a self-sheathing flow from a single input solution should have general utility for other cytometry systems and the pinched-flow design enables an order of magnitude higher throughput (65,000 cells s(-1)) compared to our previously reported deformability cytometry method, which will be especially useful for identification of rare cell populations in clinical body fluids in the future.

Method for autologous single skin cell isolation for regenerative cell spray transplantation with non-cultured cells.

PubMed

Gerlach, Jörg C; Johnen, Christa; Ottomann, Christian; Ottoman, Christian; Bräutigam, Kirsten; Plettig, Jörn; Belfekroun, Claudia; Münch, Sandra; Hartmann, Bernd

2011-03-01

There is a therapeutic gap for patients with deep partial thickness wounds (Grade IIb) of moderate size that were initially not treated with split- or mesh grafting to avoid overgrafting, but developed delayed wound healing around two weeks after injury--at which time grafting is typically not indicated anymore. Delayed wound healing is often associated with esthetically unsatisfactory results and sometimes functional problems. An innovative cell isolation method for cell spray transplantation at the point of care, which eliminates cell culture prior to treatment, was implemented for this population of burn patients in our center. Autologous skin cell spray transplantation was initiated by taking healthy skin. The dermal/epidermal layers were separated using enzymatic digestion with 40 min dispase application, followed by 15 min trypsin application for basal kerationcyte isolation, 7 min cell washing by centrifugation, followed by transferring the cells for spraying into Ringer lactate solution. The procedure was performed on site in a single session immediately following the biopsy. After sharp wound debridement, cells were immediately transplanted by deposition with a cell sprayer for even distribution of the cell suspension. Eight patients were treated (mean age 30.3 years, mean burn total body surface area 14%, mean Abbreviated Burn Severity Index (5 points). The mean time to complete re-epithelialization was 12.6 days. All patients exhibited wound healing with improved esthetic and functional quality. Our initial experience for the use of non-cultured cells using a two-enzyme approach with cell washing suggests shortened time for wound closure, suggesting that the method may potentially avoid longer-term complications.

Toward an understanding of immune cell sociology: real-time monitoring of cytokine secretion at the single-cell level.

PubMed

Shirasaki, Yoshitaka; Yamagishi, Mai; Shimura, Nanako; Hijikata, Atsushi; Ohara, Osamu

2013-01-01

The immune system is a very complex and dynamic cellular system, and its intricacies are considered akin to those of human society. Disturbance of homeostasis of the immune system results in various types of diseases; therefore, the homeostatic mechanism of the immune system has long been a subject of great interest in biology, and a lot of information has been accumulated at the cellular and the molecular levels. However, the sociological aspects of the immune system remain too abstract to address because of its high complexity, which mainly originates from a large number and variety of cell-cell interactions. As long-range interactions mediated by cytokines play a key role in the homeostasis of the immune system, cytokine secretion analyses, ranging from analyses of the micro level of individual cells to the macro level of a bulk of cell ensembles, provide us with a solid basis of a sociological viewpoint of the immune system. In this review, as the first step toward a comprehensive understanding of immune cell sociology, cytokine secretion of immune cells is surveyed with a special emphasis on the single-cell level, which has been overlooked but should serve as a basis of immune cell sociology. Now that it has become evident that large cell-to-cell variations in cytokine secretion exist at the single-cell level, we face a tricky yet interesting question: How is homeostasis maintained when the system is composed of intrinsically noisy agents? In this context, we discuss how the heterogeneity of cytokine secretion at the single-cell level affects our view of immune cell sociology. While the apparent inconsistency between homeostasis and cell-to-cell heterogeneity is difficult to address by a conventional reductive approach, comparison and integration of single-cell data with macroscopic data will offer us a new direction for the comprehensive understanding of immune cell sociology. Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.

Superresolution imaging in live Caulobacter crescentus cells using photoswitchable enhanced yellow fluorescent protein

NASA Astrophysics Data System (ADS)

Biteen, Julie S.; Thompson, Michael A.; Tselentis, Nicole K.; Shapiro, Lucy; Moerner, W. E.

2009-02-01

Recently, photoactivation and photoswitching were used to control single-molecule fluorescent labels and produce images of cellular structures beyond the optical diffraction limit (e.g., PALM, FPALM, and STORM). While previous live-cell studies relied on sophisticated photoactivatable fluorescent proteins, we show in the present work that superresolution imaging can be performed with fusions to the commonly used fluorescent protein EYFP. Rather than being photoactivated, however, EYFP can be reactivated with violet light after apparent photobleaching. In each cycle after initial imaging, only a sparse subset fluorophores is reactivated and localized, and the final image is then generated from the measured single-molecule positions. Because these methods are based on the imaging nanometer-sized single-molecule emitters and on the use of an active control mechanism to produce sparse sub-ensembles, we suggest the phrase "Single-Molecule Active-Control Microscopy" (SMACM) as an inclusive term for this general imaging strategy. In this paper, we address limitations arising from physiologically imposed upper boundaries on the fluorophore concentration by employing dark time-lapse periods to allow single-molecule motions to fill in filamentous structures, increasing the effective labeling concentration while localizing each emitter at most once per resolution-limited spot. We image cell-cycle-dependent superstructures of the bacterial actin protein MreB in live Caulobacter crescentus cells with sub-40-nm resolution for the first time. Furthermore, we quantify the reactivation quantum yield of EYFP, and find this to be 1.6 x 10-6, on par with conventional photoswitchable fluorescent proteins like Dronpa. These studies show that EYFP is a useful emitter for in vivo superresolution imaging of intracellular structures in bacterial cells.

Substoichiometric hydroxynonenylation of a single protein recapitulates whole-cell-stimulated antioxidant response.

PubMed

Parvez, Saba; Fu, Yuan; Li, Jiayang; Long, Marcus J C; Lin, Hong-Yu; Lee, Dustin K; Hu, Gene S; Aye, Yimon

2015-01-14

Lipid-derived electrophiles (LDEs) that can directly modify proteins have emerged as important small-molecule cues in cellular decision-making. However, because these diffusible LDEs can modify many targets [e.g., >700 cysteines are modified by the well-known LDE 4-hydroxynonenal (HNE)], establishing the functional consequences of LDE modification on individual targets remains devilishly difficult. Whether LDE modifications on a single protein are biologically sufficient to activate discrete redox signaling response downstream also remains untested. Herein, using T-REX (targetable reactive electrophiles and oxidants), an approach aimed at selectively flipping a single redox switch in cells at a precise time, we show that a modest level (∼34%) of HNEylation on a single target is sufficient to elicit the pharmaceutically important antioxidant response element (ARE) activation, and the resultant strength of ARE induction recapitulates that observed from whole-cell electrophilic perturbation. These data provide the first evidence that single-target LDE modifications are important individual events in mammalian physiology.

Highly sensitive detection of mutations in CHO cell recombinant DNA using multi-parallel single molecule real-time DNA sequencing.

PubMed

Cartwright, Joseph F; Anderson, Karin; Longworth, Joseph; Lobb, Philip; James, David C

2018-06-01

High-fidelity replication of biologic-encoding recombinant DNA sequences by engineered mammalian cell cultures is an essential pre-requisite for the development of stable cell lines for the production of biotherapeutics. However, immortalized mammalian cells characteristically exhibit an increased point mutation frequency compared to mammalian cells in vivo, both across their genomes and at specific loci (hotspots). Thus unforeseen mutations in recombinant DNA sequences can arise and be maintained within producer cell populations. These may affect both the stability of recombinant gene expression and give rise to protein sequence variants with variable bioactivity and immunogenicity. Rigorous quantitative assessment of recombinant DNA integrity should therefore form part of the cell line development process and be an essential quality assurance metric for instances where synthetic/multi-component assemblies are utilized to engineer mammalian cells, such as the assessment of recombinant DNA fidelity or the mutability of single-site integration target loci. Based on Pacific Biosciences (Menlo Park, CA) single molecule real-time (SMRT™) circular consensus sequencing (CCS) technology we developed a rDNA sequence analysis tool to process the multi-parallel sequencing of ∼40,000 single recombinant DNA molecules. After statistical filtering of raw sequencing data, we show that this analytical method is capable of detecting single point mutations in rDNA to a minimum single mutation frequency of 0.0042% (<1/24,000 bases). Using a stable CHO transfectant pool harboring a randomly integrated 5 kB plasmid construct encoding GFP we found that 28% of recombinant plasmid copies contained at least one low frequency (<0.3%) point mutation. These mutations were predominantly found in GC base pairs (85%) and that there was no positional bias in mutation across the plasmid sequence. There was no discernable difference between the mutation frequencies of coding and non-coding DNA. The putative ratio of non-synonymous and synonymous changes within the open reading frames (ORFs) in the plasmid sequence indicates that natural selection does not impact upon the prevalence of these mutations. Here we have demonstrated the abundance of mutations that fall outside of the reported range of detection of next generation sequencing (NGS) and second generation sequencing (SGS) platforms, providing a methodology capable of being utilized in cell line development platforms to identify the fidelity of recombinant genes throughout the production process. © 2018 Wiley Periodicals, Inc.

Controlled permeation of cell membrane by single bubble acoustic cavitation

PubMed Central

Zhou, Y.; Yang, K.; Cui, J.; Ye, J. Y.; Deng, C. X.

2011-01-01

Sonoporation is the membrane disruption generated by ultrasound and has been exploited as a non-viral strategy for drug and gene delivery. Acoustic cavitation of microbubbles has been recognized to play an important role in sonoporation. However, due to the lack of adequate techniques for precise control of cavitation activities and real-time assessment of the resulting sub-micron process of sonoporation, limited knowledge has been available regarding the detail processes and correlation of cavitation with membrane disruption at the single cell level. In the current study, we developed a combined approach including optical, acoustic, and electrophysiological techniques to enable synchronized manipulation, imaging, and measurement of cavitation of single bubbles and the resulting cell membrane disruption in real-time. Using a self-focused femtosecond laser and high frequency (7.44 MHz) pulses, a single microbubble was generated and positioned at a desired distance from the membrane of a Xenopus oocyte. Cavitation of the bubble was achieved by applying a low frequency (1.5 MHz) ultrasound pulse (duration 13.3 or 40 µs) to induce bubble collapse. Disruption of the cell membrane was assessed by the increase in the transmembrane current (TMC) of the cell under voltage clamp. Simultaneous high-speed bright field imaging of cavitation and measurements of the TMC were obtained to correlate the ultrasound-generated bubble activities with the cell membrane poration. The change in membrane permeability was directly associated with the formation of a sub-micrometer pore from a local membrane rupture generated by bubble collapse or bubble compression depending on ultrasound amplitude and duration. The impact of the bubble collapse on membrane permeation decreased rapidly with increasing distance (D) between the bubble (diameter d) and the cell membrane. The effective range of cavitation impact on membrane poration was determined to be D/d = 0.75. The maximum mean radius of the pores was estimated from the measured TMC to be 0.106 ± 0.032 µm (n = 70) for acoustic pressure of 1.5 MPa (duration 13.3 µs), and increased to 0.171 ± 0.030 µm (n = 125) for acoustic pressure of 1.7 MPa and to 0.182 ± 0.052 µm (n=112) for a pulse duration of 40 µs (1.5 MPa). These results from controlled cell membrane permeation by cavitation of single bubbles revealed insights and key factors affecting sonoporation at the single cell level. PMID:21945682

A map of protein dynamics during cell-cycle progression and cell-cycle exit

PubMed Central

Gookin, Sara; Min, Mingwei; Phadke, Harsha; Chung, Mingyu; Moser, Justin; Miller, Iain; Carter, Dylan

2017-01-01

The cell-cycle field has identified the core regulators that drive the cell cycle, but we do not have a clear map of the dynamics of these regulators during cell-cycle progression versus cell-cycle exit. Here we use single-cell time-lapse microscopy of Cyclin-Dependent Kinase 2 (CDK2) activity followed by endpoint immunofluorescence and computational cell synchronization to determine the temporal dynamics of key cell-cycle proteins in asynchronously cycling human cells. We identify several unexpected patterns for core cell-cycle proteins in actively proliferating (CDK2-increasing) versus spontaneously quiescent (CDK2-low) cells, including Cyclin D1, the levels of which we find to be higher in spontaneously quiescent versus proliferating cells. We also identify proteins with concentrations that steadily increase or decrease the longer cells are in quiescence, suggesting the existence of a continuum of quiescence depths. Our single-cell measurements thus provide a rich resource for the field by characterizing protein dynamics during proliferation versus quiescence. PMID:28892491

Peering into Cells One Molecule at a Time: Single-molecule and plasmon-enhanced fluorescence super-resolution imaging

NASA Astrophysics Data System (ADS)

Biteen, Julie

2013-03-01

Single-molecule fluorescence brings the resolution of optical microscopy down to the nanometer scale, allowing us to unlock the mysteries of how biomolecules work together to achieve the complexity that is a cell. This high-resolution, non-destructive method for examining subcellular events has opened up an exciting new frontier: the study of macromolecular localization and dynamics in living cells. We have developed methods for single-molecule investigations of live bacterial cells, and have used these techniques to investigate thee important prokaryotic systems: membrane-bound transcription activation in Vibrio cholerae, carbohydrate catabolism in Bacteroides thetaiotaomicron, and DNA mismatch repair in Bacillus subtilis. Each system presents unique challenges, and we will discuss the important methods developed for each system. Furthermore, we use the plasmon modes of bio-compatible metal nanoparticles to enhance the emissivity of single-molecule fluorophores. The resolution of single-molecule imaging in cells is generally limited to 20-40 nm, far worse than the 1.5-nm localization accuracies which have been attained in vitro. We use plasmonics to improve the brightness and stability of single-molecule probes, and in particular fluorescent proteins, which are widely used for bio-imaging. We find that gold-coupled fluorophores demonstrate brighter, longer-lived emission, yielding an overall enhancement in total photons detected. Ultimately, this results in increased localization accuracy for single-molecule imaging. Furthermore, since fluorescence intensity is proportional to local electromagnetic field intensity, these changes in decay intensity and rate serve as a nm-scale read-out of the field intensity. Our work indicates that plasmonic substrates are uniquely advantageous for super-resolution imaging, and that plasmon-enhanced imaging is a promising technique for improving live cell single-molecule microscopy.

Pinus Monophylla (Single Needled Pinyon Pine) show morphological changes in needle cell size and stomata over the past 100 years of rising CO2 in Western Arid Ecosystems.

NASA Astrophysics Data System (ADS)

Van De Water, P. K.

2016-12-01

The size, frequency, and morphology of leaf surface stomata is used to reconstruct past levels of atmospheric carbon dioxide over geologic time. This technique relies on measuring cell and cell-clusters to correlate with changes of known carbon dioxide levels in the atmosphere. Unfortunately, not all plants are suitable because the occurrence and placement of stomatal cell-complexes differ significantly between plant families. Monocot and dicot angiosperms exhibit different types of stomata and stomatal complexes that lack order and thus are unsuitable. But, in gymnosperms, the number and distribution of stomata and pavement cells is formalized and can be used to reconstruct past atmospheric carbon dioxide levels. However, characteristic of each plant species must still be considered. For example, conifers are useful but are divided into two-needle to five-needle pines, or have irregular surface morphology (Pseudotsuga sp. and Tsuga sp. needles). This study uses Pinus monophylla an undivided needle morphology, that being a cylinder has no interior surface cells. Pinus monophylla (single needle pinyon) needles were collected along Geiger Grade (Nevada State Highway 341, Reno) in 2005 and 2013 from 1500m to 2195m. Herbarium samples were also collected from 13 historic collections made between 1911 and 1994. The study determined changes with elevation and/or over time using in these populations. Using Pinus monophylla, insured needles represented a single surface with stomata, stomatal complex cells, and co-occurring pavement cell types. Results show decreased stomatal densities (stomata/area), stomatal index (stomata/stomata + epidermal cells) and stable stomata per row (stomata/row) . Epidermal cell density (Epidermal Cells /Area), and Pavement cell density (Pavement cell/area) track stomatal density similarly. Data comparison, using elevation in the 2005 and 2013 collections showed no-significant trends. Individual stomatal complexes show no differences in the size and shape over time or with elevation. Stomata morphology and the stomatal pores appear conservative. However some complex cells show a morphology suggesting they are not fully formed and functional. These characteristics appear often in the modern material suggesting some stomata never fully develop.

Injury-induced inflammation and inadequate HSP expression in mesothelial cells upon repeat exposure to dual-chamber bag peritoneal dialysis fluids.

PubMed

Bender, Thorsten O; Kratochwill, Klaus; Herzog, Rebecca; Ulbrich, Andrea; Böhm, Michael; Jörres, Achim; Aufricht, Christoph

2015-10-01

Peritoneal dialysis fluids (PDFs) may induce inadequate heat-shock protein (HSP) expression and injury-related inflammation in exposed mesothelial cells. The aim of this study was to relate cellular injury to these cellular responses in mesothelial cells following repeated exposure to 3 commercial PDFs with different biocompatibility profiles. Primary cultures of human peritoneal mesothelial cells (HPMC) were exposed to a 1:2 mixture of cell culture medium and CAPD2 (single-chamber bag PDF; Fresenius, Bad Homburg, Germany), Physioneal (dual-chamber bag PDF; Baxter, Deerfield, IL, USA) or Balance (dual-chamber bag PDF, Fresenius) for up to 10 days exposure time (4 dwells). Supernatant was analyzed for LDH, IL-6, and IL-8, cells for HSP-72 expression, and protein content. PDF exposure resulted in a biphasic pattern of cell damage switching from an earlier phase with increased injury by single-chamber PDF to a delayed phase with increased susceptibility to dual-chamber PDF. Sterile inflammation was related to LDH release over time and could be reproduced by exposure to necrotic cellular material. PDF exposure resulted in low HSP-72 expression in all tested PDFs. Exposure to single-chamber as well as to dual-chamber bag PDFs induce increased vulnerability of mesothelial cells to repeated exposure of the same solution. These effects were delayed with dual-chamber PDFs. Injury-induced inflammation and impaired HSP expression upon PDF exposure might initiate a vicious cycle with progredient mesothelial cell damage upon repeated PDF exposure. Certainly, interventional studies and translation of these results into the in vivo system is needed.

Biocompatible fluorescent silicon nanocrystals for single-molecule tracking and fluorescence imaging

PubMed Central

Nishimura, Hirohito; Ritchie, Ken; Kasai, Rinshi S.; Goto, Miki; Morone, Nobuhiro; Sugimura, Hiroyuki; Tanaka, Koichiro; Sase, Ichiro; Yoshimura, Akihiko; Nakano, Yoshitaro; Fujiwara, Takahiro K.

2013-01-01

Fluorescence microscopy is used extensively in cell-biological and biomedical research, but it is often plagued by three major problems with the presently available fluorescent probes: photobleaching, blinking, and large size. We have addressed these problems, with special attention to single-molecule imaging, by developing biocompatible, red-emitting silicon nanocrystals (SiNCs) with a 4.1-nm hydrodynamic diameter. Methods for producing SiNCs by simple chemical etching, for hydrophilically coating them, and for conjugating them to biomolecules precisely at a 1:1 ratio have been developed. Single SiNCs neither blinked nor photobleached during a 300-min overall period observed at video rate. Single receptor molecules in the plasma membrane of living cells (using transferrin receptor) were imaged for ≥10 times longer than with other probes, making it possible for the first time to observe the internalization process of receptor molecules at the single-molecule level. Spatial variations of molecular diffusivity in the scale of 1–2 µm, i.e., a higher level of domain mosaicism in the plasma membrane, were revealed. PMID:24043702

Atomic force microscopy studies on cellular elastic and viscoelastic properties.

PubMed

Li, Mi; Liu, Lianqing; Xi, Ning; Wang, Yuechao

2018-01-01

In this work, a method based on atomic force microscopy (AFM) approach-reside-retract experiments was established to simultaneously quantify the elastic and viscoelastic properties of single cells. First, the elastic and viscoelastic properties of normal breast cells and cancerous breast cells were measured, showing significant differences in Young's modulus and relaxation times between normal and cancerous breast cells. Remarkable differences in cellular topography between normal and cancerous breast cells were also revealed by AFM imaging. Next, the elastic and viscoelasitc properties of three other types of cell lines and primary normal B lymphocytes were measured; results demonstrated the potential of cellular viscoelastic properties in complementing cellular Young's modulus for discerning different states of cells. This research provides a novel way to quantify the mechanical properties of cells by AFM, which allows investigation of the biomechanical behaviors of single cells from multiple aspects.

Electronic platform for real-time multi-parametric analysis of cellular behavior post-exposure to single-walled carbon nanotubes

PubMed Central

Eldawud, Reem; Wagner, Alixandra; Dong, Chenbo; Rojansakul, Yon; Dinu, Cerasela Zoica

2016-01-01

Single-walled carbon nanotubes (SWCNTs) implementation in a variety of biomedical applications from bioimaging, to controlled drug delivery and cellular-directed alignment for muscle myofiber fabrication, has raised awareness of their potential toxicity. Nanotubes structural aspects which resemble asbestos, as well as their ability to induce cyto and genotoxicity upon interaction with biological systems by generating reactive oxygen species or inducing membrane damage, just to name a few, have led to focused efforts aimed to assess associated risks prior their user implementation. In this study, we employed a non-invasive and real-time electric cell impedance sensing (ECIS) platform to monitor behavior of lung epithelial cells upon exposure to a library of SWCNTs with user-defined physicochemical properties. Using the natural sensitivity of the cells, we evaluated SWCNT-induced cellular changes in relation to cell attachment, cell–cell interactions and cell viability respectively. Our methods have the potential to lead to the development of standardized assays for risk assessment of other nanomaterials as well as risk differentiation based on the nanomaterials surface chemistry, purity and agglomeration state. PMID:25913448

Electron microscopy of whole cells in liquid with nanometer resolution

PubMed Central

de Jonge, N.; Peckys, D. B.; Kremers, G. J.; Piston, D. W.

2009-01-01

Single gold-tagged epidermal growth factor (EGF) molecules bound to cellular EGF receptors of fixed fibroblast cells were imaged in liquid with a scanning transmission electron microscope (STEM). The cells were placed in buffer solution in a microfluidic device with electron transparent windows inside the vacuum of the electron microscope. A spatial resolution of 4 nm and a pixel dwell time of 20 μs were obtained. The liquid layer was sufficiently thick to contain the cells with a thickness of 7 ± 1 μm. The experimental findings are consistent with a theoretical calculation. Liquid STEM is a unique approach for imaging single molecules in whole cells with significantly improved resolution and imaging speed over existing methods. PMID:19164524

In-vivo RGB marking and multicolour single-cell tracking in the adult brain

PubMed Central

Gomez-Nicola, Diego; Riecken, Kristoffer; Fehse, Boris; Perry, V. Hugh

2014-01-01

In neuroscience it is a technical challenge to identify and follow the temporal and spatial distribution of cells as they differentiate. We hypothesised that RGB marking, the tagging of individual cells with unique hues resulting from simultaneous expression of the three basic colours red, green and blue, provides a convenient toolbox for the study of the CNS anatomy at the single-cell level. Using γ-retroviral and lentiviral vector sets we describe for the first time the in-vivo multicolour RGB marking of neurons in the adult brain. RGB marking also enabled us to track the spatial and temporal fate of neural stem cells in the adult brain. The application of different viral envelopes and promoters provided a useful approach to track the generation of neurons vs. glial cells at the neurogenic niche, allowing the identification of the prominent generation of new astrocytes to the striatum. Multicolour RGB marking could serve as a universal and reproducible method to study and manipulate the CNS at the single-cell level, in both health and disease. PMID:25531807

Ciliary heterogeneity within a single cell: the Paramecium model.

PubMed

Aubusson-Fleury, Anne; Cohen, Jean; Lemullois, Michel

2015-01-01

Paramecium is a single cell able to divide in its morphologically differentiated stage that has many cilia anchored at its cell surface. Many thousands of cilia are thus assembled in a short period of time during division to duplicate the cell pattern while the cell continues swimming. Most, but not all, of these sensory cilia are motile and involved in two main functions: prey capture and cell locomotion. These cilia display heterogeneity, both in their length and their biochemical properties. Thanks to these properties, as well as to the availability of many postgenomic tools and the possibility to follow the regrowth of cilia after deciliation, Paramecium offers a nice opportunity to study the assembly of the cilia, as well as the genesis of their diversity within a single cell. In this paper, after a brief survey of Paramecium morphology and cilia properties, we describe the tools and the protocols currently used for immunofluorescence, transmission electron microscopy, and ultrastructural immunocytochemistry to analyze cilia, with special recommendations to overcome the problem raised by cilium diversity. Copyright © 2015. Published by Elsevier Inc.

A single-cell spiking model for the origin of grid-cell patterns

PubMed Central

Kempter, Richard

2017-01-01

Spatial cognition in mammals is thought to rely on the activity of grid cells in the entorhinal cortex, yet the fundamental principles underlying the origin of grid-cell firing are still debated. Grid-like patterns could emerge via Hebbian learning and neuronal adaptation, but current computational models remained too abstract to allow direct confrontation with experimental data. Here, we propose a single-cell spiking model that generates grid firing fields via spike-rate adaptation and spike-timing dependent plasticity. Through rigorous mathematical analysis applicable in the linear limit, we quantitatively predict the requirements for grid-pattern formation, and we establish a direct link to classical pattern-forming systems of the Turing type. Our study lays the groundwork for biophysically-realistic models of grid-cell activity. PMID:28968386

Visualizing interactions between Sindbis virus and cells by single particle tracking

NASA Astrophysics Data System (ADS)

Williard, Mary

2005-03-01

Sindbis virus infects both mammalian and insect cells. Though not pathogenic in humans, Sindbis is a model for many mosquito- borne viruses that cause human disease, such as West Nile virus. We have used real-time single particle fluorescence microscopy to observe individual Sindbis virus particles as they infect living cells. Fluorescent labels were incorporated into both the viral coat proteins and the lipid envelope of the virus. Kinetics characteristic of free diffusion in solution, slower diffusion inside cells, attachment to spots on the cell surface, and motor protein transport inside cells have been observed. Dequenching of the membrane label is used to report membrane fusion events during the infection process. Tracking individual viral particles allows multiple pathways to be determined without the requirement of synchronicity.

Revealing nonergodic dynamics in living cells from a single particle trajectory

NASA Astrophysics Data System (ADS)

Lanoiselée, Yann; Grebenkov, Denis S.

2016-05-01

We propose the improved ergodicity and mixing estimators to identify nonergodic dynamics from a single particle trajectory. The estimators are based on the time-averaged characteristic function of the increments and can thus capture additional information on the process as compared to the conventional time-averaged mean-square displacement. The estimators are first investigated and validated for several models of anomalous diffusion, such as ergodic fractional Brownian motion and diffusion on percolating clusters, and nonergodic continuous-time random walks and scaled Brownian motion. The estimators are then applied to two sets of earlier published trajectories of mRNA molecules inside live Escherichia coli cells and of Kv2.1 potassium channels in the plasma membrane. These statistical tests did not reveal nonergodic features in the former set, while some trajectories of the latter set could be classified as nonergodic. Time averages along such trajectories are thus not representative and may be strongly misleading. Since the estimators do not rely on ensemble averages, the nonergodic features can be revealed separately for each trajectory, providing a more flexible and reliable analysis of single-particle tracking experiments in microbiology.

Real time imaging of live cell ATP leaking or release events by chemiluminescence microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yun

The purpose of this research was to expand the chemiluminescence microscopy applications in live bacterial/mammalian cell imaging and to improve the detection sensitivity for ATP leaking or release events. We first demonstrated that chemiluminescence (CL) imaging can be used to interrogate single bacterial cells. While using a luminometer allows detecting ATP from cell lysate extracted from at least 10 bacterial cells, all previous cell CL detection never reached this sensitivity of single bacteria level. We approached this goal with a different strategy from before: instead of breaking bacterial cell membrane and trying to capture the transiently diluted ATP with themore » firefly luciferase CL assay, we introduced the firefly luciferase enzyme into bacteria using the modern genetic techniques and placed the CL reaction substrate D-luciferin outside the cells. By damaging the cell membrane with various antibacterial drugs including antibiotics such as Penicillins and bacteriophages, the D-luciferin molecules diffused inside the cell and initiated the reaction that produces CL light. As firefly luciferases are large protein molecules which are retained within the cells before the total rupture and intracellular ATP concentration is high at the millmolar level, the CL reaction of firefly luciferase, ATP and D-luciferin can be kept for a relatively long time within the cells acting as a reaction container to generate enough photons for detection by the extremely sensitive intensified charge coupled device (ICCD) camera. The result was inspiring as various single bacterium lysis and leakage events were monitored with 10-s temporal resolution movies. We also found a new way of enhancing diffusion D-luciferin into cells by dehydrating the bacteria. Then we started with this novel single bacterial CL imaging technique, and applied it for quantifying gene expression levels from individual bacterial cells. Previous published result in single cell gene expression quantification mainly used a fluorescence method; CL detection is limited because of the difficulty to introduce enough D-luciferin molecules. Since dehydration could easily cause proper size holes in bacterial cell membranes and facilitate D-luciferin diffusion, we used this method and recorded CL from individual cells each hour after induction. The CL light intensity from each individual cell was integrated and gene expression levels of two strain types were compared. Based on our calculation, the overall sensitivity of our system is already approaching the single enzyme level. The median enzyme number inside a single bacterium from the higher expression strain after 2 hours induction was quantified to be about 550 molecules. Finally we imaged ATP release from astrocyte cells. Upon mechanical stimulation, astrocyte cells respond by increasing intracellular Ca 2+ level and releasing ATP to extracellular spaces as signaling molecules. The ATP release imaged by direct CL imaging using free firefly luciferase and D-luciferin outside cells reflects the transient release as well as rapid ATP diffusion. Therefore ATP release detection at the cell surface is critical to study the ATP release mechanism and signaling propagation pathway. We realized this cell surface localized ATP release imaging detection by immobilizing firefly luciferase to streptavidin beads that attached to the cell surface via streptavidin-biotin interactions. Both intracellular Ca 2+ propagation wave and extracellular ATP propagation wave at the cell surface were recorded with fluorescence and CL respectively. The results imply that at close distances from the stimulation center (<120 μm) extracellular ATP pathway is faster, while at long distances (>120 μm) intracellular Ca 2+ signaling through gap junctions seems more effective.« less

A non-aggressive, highly efficient, enzymatic method for dissociation of human brain-tumors and brain-tissues to viable single-cells.

PubMed

Volovitz, Ilan; Shapira, Netanel; Ezer, Haim; Gafni, Aviv; Lustgarten, Merav; Alter, Tal; Ben-Horin, Idan; Barzilai, Ori; Shahar, Tal; Kanner, Andrew; Fried, Itzhak; Veshchev, Igor; Grossman, Rachel; Ram, Zvi

2016-06-01

Conducting research on the molecular biology, immunology, and physiology of brain tumors (BTs) and primary brain tissues requires the use of viably dissociated single cells. Inadequate methods for tissue dissociation generate considerable loss in the quantity of single cells produced and in the produced cells' viability. Improper dissociation may also demote the quality of data attained in functional and molecular assays due to the presence of large quantities cellular debris containing immune-activatory danger associated molecular patterns, and due to the increased quantities of degraded proteins and RNA. Over 40 resected BTs and non-tumorous brain tissue samples were dissociated into single cells by mechanical dissociation or by mechanical and enzymatic dissociation. The quality of dissociation was compared for all frequently used dissociation enzymes (collagenase, DNase, hyaluronidase, papain, dispase) and for neutral protease (NP) from Clostridium histolyticum. Single-cell-dissociated cell mixtures were evaluated for cellular viability and for the cell-mixture dissociation quality. Dissociation quality was graded by the quantity of subcellular debris, non-dissociated cell clumps, and DNA released from dead cells. Of all enzymes or enzyme combinations examined, NP (an enzyme previously not evaluated on brain tissues) produced dissociated cell mixtures with the highest mean cellular viability: 93 % in gliomas, 85 % in brain metastases, and 89 % in non-tumorous brain tissue. NP also produced cell mixtures with significantly less cellular debris than other enzymes tested. Dissociation using NP was non-aggressive over time-no changes in cell viability or dissociation quality were found when comparing 2-h dissociation at 37 °C to overnight dissociation at ambient temperature. The use of NP allows for the most effective dissociation of viable single cells from human BTs or brain tissue. Its non-aggressive dissociative capacity may enable ambient-temperature shipping of tumor pieces in multi-center clinical trials, meanwhile being dissociated. As clinical grade NP is commercially available it can be easily integrated into cell-therapy clinical trials in neuro-oncology. The high quality viable cells produced may enable investigators to conduct more consistent research by avoiding the experimental artifacts associated with the presence dead cells or cellular debris.

The slow cell death response when screening chemotherapeutic agents.

PubMed

Blois, Joseph; Smith, Adam; Josephson, Lee

2011-09-01

To examine the correlation between cell death and a common surrogate of death used in screening assays, we compared cell death responses to those obtained with the sulforhodamine B (SRB) cell protein-based "cytotoxicity" assay. With the SRB assay, the Hill equation was used to obtain an IC50 and final cell mass, or cell mass present at infinite agent concentrations, with eight adherent cell lines and four agents (32 agent/cell combinations). Cells were treated with high agent concentrations (well above the SRB IC50) and the death response determined as the time-dependent decrease in cells failing to bind both annexin V and vital fluorochromes by flow cytometry. Death kinetics were categorized as fast (5/32) (similar to the reference nonadherent Jurkat line), slow (17/32), or none (10/32), despite positive responses in the SRB assay in all cases. With slow cell death, a single exposure to a chemotherapeutic agent caused a slow, progressive increase in dead (necrotic) and dying (apoptotic) cells for at least 72 h. Cell death (defined by annexin and/or fluorochrome binding) did not correlate with the standard SRB "cytotoxicity" assay. With the slow cell death response, a single exposure to an agent caused a slow conversion from vital to apoptotic and necrotic cells over at least 72 h (the longest time point examined). Here, increasing the time of exposure to agent concentrations modestly above the SRB IC50 provides a method of maximizing cell kill. If tumors respond similarly, sustained low doses of chemotherapeutic agents, rather than a log-kill, maximum tolerated dose strategy may be an optimal strategy of maximizing tumor cell death.

Mapping power-law rheology of living cells using multi-frequency force modulation atomic force microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takahashi, Ryosuke; Okajima, Takaharu, E-mail: okajima@ist.hokudai.ac.jp

We present multi-frequency force modulation atomic force microscopy (AFM) for mapping the complex shear modulus G* of living cells as a function of frequency over the range of 50–500 Hz in the same measurement time as the single-frequency force modulation measurement. The AFM technique enables us to reconstruct image maps of rheological parameters, which exhibit a frequency-dependent power-law behavior with respect to G{sup *}. These quantitative rheological measurements reveal a large spatial variation in G* in this frequency range for single cells. Moreover, we find that the reconstructed images of the power-law rheological parameters are much different from those obtained inmore » force-curve or single-frequency force modulation measurements. This indicates that the former provide information about intracellular mechanical structures of the cells that are usually not resolved with the conventional force measurement methods.« less

Single cell analysis of voltage-gated potassium channels that determines neuronal types of rat hypothalamic paraventricular nucleus neurons.

PubMed

Lee, S K; Lee, S; Shin, S Y; Ryu, P D; Lee, S Y

2012-03-15

The hypothalamic paraventricular nucleus (PVN), a site for the integration of both the neuroendocrine and autonomic systems, has heterogeneous cell composition. These neurons are classified into type I and type II neurons based on their electrophysiological properties. In the present study, we investigated the molecular identification of voltage-gated K+ (Kv) channels, which determines a distinctive characteristic of type I PVN neurons, by means of single-cell reverse transcription-polymerase chain reaction (RT-PCR) along with slice patch clamp recordings. In order to determine the mRNA expression profiles, firstly, the PVN neurons of male rats were classified into type I and type II neurons, and then, single-cell RT-PCR and single-cell real-time RT-PCR analysis were performed using the identical cell. The single-cell RT-PCR analysis revealed that Kv1.2, Kv1.3, Kv1.4, Kv4.1, Kv4.2, and Kv4.3 were expressed both in type I and in type II neurons, and several Kv channels were co-expressed in a single PVN neuron. However, we found that the expression densities of Kv4.2 and Kv4.3 were significantly higher in type I neurons than in type II neurons. Taken together, several Kv channels encoding A-type K+ currents are present both in type I and in type II neurons, and among those, Kv4.2 and Kv4.3 are the major Kv subunits responsible for determining the distinct electrophysiological properties. Thus these 2 Kv subunits may play important roles in determining PVN cell types and regulating PVN neuronal excitability. This study further provides key molecular mechanisms for differentiating type I and type II PVN neurons. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Conditional Dispersive Readout of a CMOS Single-Electron Memory Cell

NASA Astrophysics Data System (ADS)

Schaal, S.; Barraud, S.; Morton, J. J. L.; Gonzalez-Zalba, M. F.

2018-05-01

Quantum computers require interfaces with classical electronics for efficient qubit control, measurement, and fast data processing. Fabricating the qubit and the classical control layer using the same technology is appealing because it will facilitate the integration process, improving feedback speeds and offering potential solutions to wiring and layout challenges. Integrating classical and quantum devices monolithically, using complementary metal-oxide-semiconductor (CMOS) processes, enables the processor to profit from the most mature industrial technology for the fabrication of large-scale circuits. We demonstrate a CMOS single-electron memory cell composed of a single quantum dot and a transistor that locks charge on the quantum-dot gate. The single-electron memory cell is conditionally read out by gate-based dispersive sensing using a lumped-element L C resonator. The control field-effect transistor (FET) and quantum dot are fabricated on the same chip using fully depleted silicon-on-insulator technology. We obtain a charge sensitivity of δ q =95 ×10-6e Hz-1 /2 when the quantum-dot readout is enabled by the control FET, comparable to results without the control FET. Additionally, we observe a single-electron retention time on the order of a second when storing a single-electron charge on the quantum dot at millikelvin temperatures. These results demonstrate first steps towards time-based multiplexing of gate-based dispersive readout in CMOS quantum devices opening the path for the development of an all-silicon quantum-classical processor.

Human neuronal changes in brain edema and increased intracranial pressure.

PubMed

Faragó, Nóra; Kocsis, Ágnes Katalin; Braskó, Csilla; Lovas, Sándor; Rózsa, Márton; Baka, Judith; Kovács, Balázs; Mikite, Katalin; Szemenyei, Viktor; Molnár, Gábor; Ozsvár, Attila; Oláh, Gáspár; Piszár, Ildikó; Zvara, Ágnes; Patócs, Attila; Barzó, Pál; Puskás, László G; Tamás, Gábor

2016-08-04

Functional and molecular changes associated with pathophysiological conditions are relatively easily detected based on tissue samples collected from patients. Population specific cellular responses to disease might remain undiscovered in samples taken from organs formed by a multitude of cell types. This is particularly apparent in the human cerebral cortex composed of a yet undefined number of neuron types with a potentially different involvement in disease processes. We combined cellular electrophysiology, anatomy and single cell digital PCR in human neurons identified in situ for the first time to assess mRNA expression and corresponding functional changes in response to edema and increased intracranial pressure. In single pyramidal cells, mRNA copy numbers of AQP1, AQP3, HMOX1, KCNN4, SCN3B and SOD2 increased, while CACNA1B, CRH decreased in edema. In addition, single pyramidal cells increased the copy number of AQP1, HTR5A and KCNS1 mRNAs in response to increased intracranial pressure. In contrast to pyramidal cells, AQP1, HMOX1and KCNN4 remained unchanged in single cell digital PCR performed on fast spiking cells in edema. Corroborating single cell digital PCR results, pharmacological and immunohistochemical results also suggested the presence of KCNN4 encoding the α-subunit of KCa3.1 channels in edema on pyramidal cells, but not on interneurons. We measured the frequency of spontaneous EPSPs on pyramidal cells in both pathophysiological conditions and on fast spiking interneurons in edema and found a significant decrease in each case, which was accompanied by an increase in input resistances on both cell types and by a drop in dendritic spine density on pyramidal cells consistent with a loss of excitatory synapses. Our results identify anatomical and/or physiological changes in human pyramidal and fast spiking cells in edema and increased intracranial pressure revealing cell type specific quantitative changes in gene expression. Some of the edema/increased intracranial pressure modulated and single human pyramidal cell verified gene products identified here might be considered as novel pharmacological targets in cell type specific neuroprotection.

Frequent pauses in Escherichia coli flagella elongation revealed by single cell real-time fluorescence imaging.

PubMed

Zhao, Ziyi; Zhao, Yifan; Zhuang, Xiang-Yu; Lo, Wei-Chang; Baker, Matthew A B; Lo, Chien-Jung; Bai, Fan

2018-05-14

The bacterial flagellum is a large extracellular protein organelle that extrudes from the cell surface. The flagellar filament is assembled from tens of thousands of flagellin subunits that are exported through the flagellar type III secretion system. Here, we measure the growth of Escherichia coli flagella in real time and find that, although the growth rate displays large variations at similar lengths, it decays on average as flagella lengthen. By tracking single flagella, we show that the large variations in growth rate occur as a result of frequent pauses. Furthermore, different flagella on the same cell show variable growth rates with correlation. Our observations are consistent with an injection-diffusion model, and we propose that an insufficient cytoplasmic flagellin supply is responsible for the pauses in flagellar growth in E. coli.

Investigating cell-substrate and cell-cell interactions by means of single-cell-probe force spectroscopy.

PubMed

Moreno-Cencerrado, Alberto; Iturri, Jagoba; Pecorari, Ilaria; D M Vivanco, Maria; Sbaizero, Orfeo; Toca-Herrera, José L

2017-01-01

Cell adhesion forces are typically a mixture of specific and nonspecific cell-substrate and cell-cell interactions. In order to resolve these phenomena, Atomic Force Microscopy appears as a powerful device which can measure cell parameters by means of manipulation of single cells. This method, commonly known as cell-probe force spectroscopy, allows us to control the force applied, the area of interest, the approach/retracting speed, the force rate, and the time of interaction. Here, we developed a novel approach for in situ cantilever cell capturing and measurement of specific cell interactions. In particular, we present a new setup consisting of two different half-surfaces coated either with recrystallized SbpA bacterial cell surface layer proteins (S-layers) or integrin binding Fibronectin, on which MCF-7 breast cancer cells are incubated. The presence of a clear physical boundary between both surfaces benefits for a quick detection of the region under analysis. Thus, quantitative results about SbpA-cell and Fibronectin-cell adhesion forces as a function of the contact time are described. Additionally, the importance of the cell spreading in cell-cell interactions has been studied for surfaces coated with two different Fibronectin concentrations: 20 μg/mL (FN20) and 100 μg/mL (FN100), which impact the number of substrate receptors. Microsc. Res. Tech. 80:124-130, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Exploring the Underlying Mechanisms of the Xenopus laevis Embryonic Cell Cycle.

PubMed

Zhang, Kun; Wang, Jin

2018-05-31

The cell cycle is an indispensable process in proliferation and development. Despite significant efforts, global quantification and physical understanding are still challenging. In this study, we explored the mechanisms of the Xenopus laevis embryonic cell cycle by quantifying the underlying landscape and flux. We uncovered the Mexican hat landscape of the Xenopus laevis embryonic cell cycle with several local basins and barriers on the oscillation path. The local basins characterize the different phases of the Xenopus laevis embryonic cell cycle, and the local barriers represent the checkpoints. The checkpoint mechanism of the cell cycle is revealed by the landscape basins and barriers. While landscape shape determines the stabilities of the states on the oscillation path, the curl flux force determines the stability of the cell cycle flow. Replication is fundamental for biology of living cells. We quantify the input energy (through the entropy production) as the thermodynamic requirement for initiation and sustainability of single cell life (cell cycle). Furthermore, we also quantify curl flux originated from the input energy as the dynamical requirement for the emergence of a new stable phase (cell cycle). This can provide a new quantitative insight for the origin of single cell life. In fact, the curl flux originated from the energy input or nutrition supply determines the speed and guarantees the progression of the cell cycle. The speed of the cell cycle is a hallmark of cancer. We characterized the quality of the cell cycle by the coherence time and found it is supported by the flux and energy cost. We are also able to quantify the degree of time irreversibility by the cross correlation function forward and backward in time from the stochastic traces in the simulation or experiments, providing a way for the quantification of the time irreversibility and the flux. Through global sensitivity analysis upon landscape and flux, we can identify the key elements for controlling the cell cycle speed. This can help to design an effective strategy for drug discovery against cancer.

Extracting Cell Stiffness from Real-Time Deformability Cytometry: Theory and Experiment.

PubMed

Mietke, Alexander; Otto, Oliver; Girardo, Salvatore; Rosendahl, Philipp; Taubenberger, Anna; Golfier, Stefan; Ulbricht, Elke; Aland, Sebastian; Guck, Jochen; Fischer-Friedrich, Elisabeth

2015-11-17

Cell stiffness is a sensitive indicator of physiological and pathological changes in cells, with many potential applications in biology and medicine. A new method, real-time deformability cytometry, probes cell stiffness at high throughput by exposing cells to a shear flow in a microfluidic channel, allowing for mechanical phenotyping based on single-cell deformability. However, observed deformations of cells in the channel not only are determined by cell stiffness, but also depend on cell size relative to channel size. Here, we disentangle mutual contributions of cell size and cell stiffness to cell deformation by a theoretical analysis in terms of hydrodynamics and linear elasticity theory. Performing real-time deformability cytometry experiments on both model spheres of known elasticity and biological cells, we demonstrate that our analytical model not only predicts deformed shapes inside the channel but also allows for quantification of cell mechanical parameters. Thereby, fast and quantitative mechanical sampling of large cell populations becomes feasible. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Extracting Cell Stiffness from Real-Time Deformability Cytometry: Theory and Experiment

PubMed Central

Mietke, Alexander; Otto, Oliver; Girardo, Salvatore; Rosendahl, Philipp; Taubenberger, Anna; Golfier, Stefan; Ulbricht, Elke; Aland, Sebastian; Guck, Jochen; Fischer-Friedrich, Elisabeth

2015-01-01

Cell stiffness is a sensitive indicator of physiological and pathological changes in cells, with many potential applications in biology and medicine. A new method, real-time deformability cytometry, probes cell stiffness at high throughput by exposing cells to a shear flow in a microfluidic channel, allowing for mechanical phenotyping based on single-cell deformability. However, observed deformations of cells in the channel not only are determined by cell stiffness, but also depend on cell size relative to channel size. Here, we disentangle mutual contributions of cell size and cell stiffness to cell deformation by a theoretical analysis in terms of hydrodynamics and linear elasticity theory. Performing real-time deformability cytometry experiments on both model spheres of known elasticity and biological cells, we demonstrate that our analytical model not only predicts deformed shapes inside the channel but also allows for quantification of cell mechanical parameters. Thereby, fast and quantitative mechanical sampling of large cell populations becomes feasible. PMID:26588562

Imaging tumor microscopic viscosity in vivo using molecular rotors

PubMed Central

Shimolina, Lyubov’ E.; Izquierdo, Maria Angeles; López-Duarte, Ismael; Bull, James A.; Shirmanova, Marina V.; Klapshina, Larisa G.; Zagaynova, Elena V.; Kuimova, Marina K.

2017-01-01

The microscopic viscosity plays an essential role in cellular biophysics by controlling the rates of diffusion and bimolecular reactions within the cell interior. While several approaches have emerged that have allowed the measurement of viscosity and diffusion on a single cell level in vitro, the in vivo viscosity monitoring has not yet been realized. Here we report the use of fluorescent molecular rotors in combination with Fluorescence Lifetime Imaging Microscopy (FLIM) to image microscopic viscosity in vivo, both on a single cell level and in connecting tissues of subcutaneous tumors in mice. We find that viscosities recorded from single tumor cells in vivo correlate well with the in vitro values from the same cancer cell line. Importantly, our new method allows both imaging and dynamic monitoring of viscosity changes in real time in live animals and thus it is particularly suitable for diagnostics and monitoring of the progress of treatments that might be accompanied by changes in microscopic viscosity. PMID:28134273

Imaging trace element distributions in single organelles and subcellular features

DOE PAGES

Kashiv, Yoav; Austin, Jotham R.; Lai, Barry; ...

2016-02-25

The distributions of chemical elements within cells are of prime importance in a wide range of basic and applied biochemical research. An example is the role of the subcellular Zn distribution in Zn homeostasis in insulin producing pancreatic beta cells and the development of type 2 diabetes mellitus. We combined transmission electron microscopy with micro-and nano-synchrotron X-ray fluorescence to image unequivocally for the first time, to the best of our knowledge, the natural elemental distributions, including those of trace elements, in single organelles and other subcellular features. Detected elements include Cl, K, Ca, Co, Ni, Cu, Zn and Cd (whichmore » some cells were supplemented with). Cell samples were prepared by a technique that minimally affects the natural elemental concentrations and distributions, and without using fluorescent indicators. In conclusion, it could likely be applied to all cell types and provide new biochemical insights at the single organelle level not available from organelle population level studies.« less

Quantification of multiple gene expression in individual cells.

PubMed

Peixoto, António; Monteiro, Marta; Rocha, Benedita; Veiga-Fernandes, Henrique

2004-10-01

Quantitative gene expression analysis aims to define the gene expression patterns determining cell behavior. So far, these assessments can only be performed at the population level. Therefore, they determine the average gene expression within a population, overlooking possible cell-to-cell heterogeneity that could lead to different cell behaviors/cell fates. Understanding individual cell behavior requires multiple gene expression analyses of single cells, and may be fundamental for the understanding of all types of biological events and/or differentiation processes. We here describe a new reverse transcription-polymerase chain reaction (RT-PCR) approach allowing the simultaneous quantification of the expression of 20 genes in the same single cell. This method has broad application, in different species and any type of gene combination. RT efficiency is evaluated. Uniform and maximized amplification conditions for all genes are provided. Abundance relationships are maintained, allowing the precise quantification of the absolute number of mRNA molecules per cell, ranging from 2 to 1.28 x 10(9) for each individual gene. We evaluated the impact of this approach on functional genetic read-outs by studying an apparently homogeneous population (monoclonal T cells recovered 4 d after antigen stimulation), using either this method or conventional real-time RT-PCR. Single-cell studies revealed considerable cell-to-cell variation: All T cells did not express all individual genes. Gene coexpression patterns were very heterogeneous. mRNA copy numbers varied between different transcripts and in different cells. As a consequence, this single-cell assay introduces new and fundamental information regarding functional genomic read-outs. By comparison, we also show that conventional quantitative assays determining population averages supply insufficient information, and may even be highly misleading.

CD8 single-cell gene coexpression reveals three different effector types present at distinct phases of the immune response

PubMed Central

Peixoto, António; Evaristo, César; Munitic, Ivana; Monteiro, Marta; Charbit, Alain; Rocha, Benedita; Veiga-Fernandes, Henrique

2007-01-01

To study in vivo CD8 T cell differentiation, we quantified the coexpression of multiple genes in single cells throughout immune responses. After in vitro activation, CD8 T cells rapidly express effector molecules and cease their expression when the antigen is removed. Gene behavior after in vivo activation, in contrast, was quite heterogeneous. Different mRNAs were induced at very different time points of the response, were transcribed during different time periods, and could decline or persist independently of the antigen load. Consequently, distinct gene coexpression patterns/different cell types were generated at the various phases of the immune responses. During primary stimulation, inflammatory molecules were induced and down-regulated shortly after activation, generating early cells that only mediated inflammation. Cytotoxic T cells were generated at the peak of the primary response, when individual cells simultaneously expressed multiple killer molecules, whereas memory cells lost killer capacity because they no longer coexpressed killer genes. Surprisingly, during secondary responses gene transcription became permanent. Secondary cells recovered after antigen elimination were more efficient killers than cytotoxic T cells present at the peak of the primary response. Thus, primary responses produced two transient effector types. However, after boosting, CD8 T cells differentiate into long-lived killer cells that persist in vivo in the absence of antigen. PMID:17485515

Small stack performance of intermediate temperature-operating solid oxide fuel cells using stainless steel interconnects and anode-supported single cell

NASA Astrophysics Data System (ADS)

Bae, Joongmyeon; Lim, Sungkwang; Jee, Hyunjin; Kim, Jung Hyun; Yoo, Young-Sung; Lee, Taehee

We are developing 1 kW class solid oxide fuel cell (SOFC) system for residential power generation (RPG) application supported by Korean Government. Anode-supported single cells with thin electrolyte layer of YSZ (yttria-stabilized zirconia) or ScSZ (scandia-stabilized zirconia) for intermediate temperature operation (650-750 °C), respectively, were fabricated and small stacks were built and evaluated. The LSCF/ScSZ/Ni-YSZ single cell showed performance of 543 mW cm -2 at 650 °C and 1680 mW cm -2 at 750 °C. The voltage of 15-cell stack based on 5 cm × 5 cm single cell (LSM/YSZ/Ni-YSZ) at 150 mW was 12.5 V in hydrogen as fuel of 120 sccm per cell at 750 °C and decreased to about 10.9 V at 500 h operation time. A 5-cell stack based on the LSCF/YSZ/FL/Ni-YSZ showed the maximum power density of 30 W, 25 W and 20 W at 750 °C, 700 °C and 650 °C, respectively. LSCF/ScSZ/Ni-YSZ-based stack showed better performance than LSCF/YSZ/Ni-YSZ stack from the experiment temperature range. I- V characteristics by using hydrogen gas and reformate gas of methane as fuel were investigated at 750 °C in LSCF/ScSZ/FL/Ni-YSZ-based 5-cell stack.

Large grained perovskite solar cells derived from single-crystal perovskite powders with enhanced ambient stability

DOE PAGES

Yen, Hung -Ju; Liang, Po -Wei; Chueh, Chu -Chen; ...

2016-05-25

In this study, we demonstrate the large grained perovskite solar cells prepared from precursor solution comprising single-crystal perovskite powders for the first time. Here, the resultant large grained perovskite thin film possesses negligible physical (structural) gap between each large grain and are highly crystalline as evidenced by its fan-shaped birefringence observed under polarized light, which is very different to the thin film prepared from the typical precursor route (MAI + PbI 2).

Probing Single- to Multi-Cell Level Charge Transport in Geobacter sulfurreducens DL-1

DTIC Science & Technology

2013-11-08

nanoelectrodes in which an array of nanoholes (200 400 nm2) precludes or single window (6 10 mm2) allows for direct microbe/electrode contacts (Supplementary...previous measurement with S. oneidensis MR-1 cells, which gave almost identical current output between window and nanohole electrodes30. At longer times, it...is interesting to note that the nanohole electrode was also able to yield a current, albeit at a much smaller magnitude, which could be attributed to

Electroporation followed by electrochemical measurement of quantal transmitter release from single cells using a patterned microelectrode.

PubMed

Ghosh, Jaya; Liu, Xin; Gillis, Kevin D

2013-06-07

An electrochemical microelectrode located immediately adjacent to a single neuroendocrine cell can record spikes of amperometric current that result from exocytosis of oxidizable transmitter from individual vesicles, i.e., quantal exocytosis. Here, we report the development of an efficient method where the same electrochemical microelectrode is used to electropermeabilize an adjacent chromaffin cell and then measure the consequent quantal catecholamine release using amperometry. Trains of voltage pulses, 5-7 V in amplitude and 0.1-0.2 ms in duration, were used to reliably trigger release from cells using gold electrodes. Amperometric spikes induced by electropermeabilization had similar areas, peak heights and durations as amperometric spikes elicited by depolarizing high K(+) solutions, therefore release occurs from individual secretory granules. Uptake of trypan blue stain into cells demonstrated that the plasma membrane is permeabilized by the voltage stimulus. Voltage pulses did not degrade the electrochemical sensitivity of the electrodes assayed using a test analyte. Surprisingly, robust quantal release was elicited upon electroporation in the absence of Ca(2+) in the bath solution (0 Ca(2+)/5 mM EGTA). In contrast, electropermeabilization-induced transmitter release required Cl(-) in the bath solution in that bracketed experiments demonstrated a steep dependence of the rate of electropermeabilization-induced transmitter release on [Cl(-)] between 2 and 32 mM. Using the same electrochemical electrode to electroporate and record quantal release of catecholamines from an individual chromaffin cell allows precise timing of the stimulus, stimulation of a single cell at a time, and can be used to load membrane-impermeant substances into a cell.

Droplet-based microfluidics platform for measurement of rapid erythrocyte water transport

PubMed Central

Jin, Byung-Ju; Esteva-Font, Cristina; Verkman, A.S.

2015-01-01

Cell membrane water permeability is an important determinant of epithelial fluid secretion, tissue swelling, angiogenesis, tumor spread and other biological processes. Cellular water channels, the aquaporins, are important drug targets. Water permeability is generally measured from the kinetics of cell volume change in response to an osmotic gradient. Here, we developed a microfluidics platform in which cells expressing a cytoplasmic, volume-sensing fluorescent dye are rapidly subjected to an osmotic gradient by solution mixing inside a ~ 0.1 nL droplet surrounded by oil. Solution mixing time was < 10 ms. Osmotic water permeability was deduced from a single, time-integrated fluorescence image of an observation area in which time after mixing is determined by spatial position. Water permeability was accurately measured in aquaporin-expressing erythrocytes with half-times for osmotic equilibration down to < 50 ms. Compared with conventional water permeability measurements using costly stopped-flow instrumentation, the microfluidics platform here utilizes sub-microliter blood sample volume, does not suffer from mixing artifact, and replaces challenging kinetic measurements by a single image capture using a standard laboratory fluorescence microscope. PMID:26159099

Menthol Suppresses Nicotinic Acetylcholine Receptor Functioning in Sensory Neurons via Allosteric Modulation

PubMed Central

Wilhelm, M.; Swandulla, D.

2012-01-01

In this study, we have investigated how the function of native and recombinant nicotinic acetylcholine receptors (nAChRs) is modulated by the monoterpenoid alcohol from peppermint (−) menthol. In trigeminal neurons (TG), we found that nicotine (75 μM)-activated whole-cell currents through nAChRs were reversibly reduced by menthol in a concentration-dependent manner with an IC50 of 111 μM. To analyze the mechanism underlying menthol's action in more detail, we used single channel and whole-cell recordings from recombinant human α4β2 nAChR expressed in HEK tsA201 cells. Here, we found a shortening of channel open time and a prolongation of channel closed time, and an increase in single channel amplitude leading in summary to a reduction in single channel current. Furthermore, menthol did not affect nicotine's EC50 value for currents through recombinant human α4β2 nAChRs but caused a significant reduction in nicotine's efficacy. Taken together, these findings indicate that menthol is a negative allosteric modulator of nAChRs. PMID:22281529

Micro-patterned agarose gel devices for single-cell high-throughput microscopy of E. coli cells.

PubMed

Priest, David G; Tanaka, Nobuyuki; Tanaka, Yo; Taniguchi, Yuichi

2017-12-21

High-throughput microscopy of bacterial cells elucidated fundamental cellular processes including cellular heterogeneity and cell division homeostasis. Polydimethylsiloxane (PDMS)-based microfluidic devices provide advantages including precise positioning of cells and throughput, however device fabrication is time-consuming and requires specialised skills. Agarose pads are a popular alternative, however cells often clump together, which hinders single cell quantitation. Here, we imprint agarose pads with micro-patterned 'capsules', to trap individual cells and 'lines', to direct cellular growth outwards in a straight line. We implement this micro-patterning into multi-pad devices called CapsuleHotel and LineHotel for high-throughput imaging. CapsuleHotel provides ~65,000 capsule structures per mm 2 that isolate individual Escherichia coli cells. In contrast, LineHotel provides ~300 line structures per mm that direct growth of micro-colonies. With CapsuleHotel, a quantitative single cell dataset of ~10,000 cells across 24 samples can be acquired and analysed in under 1 hour. LineHotel allows tracking growth of > 10 micro-colonies across 24 samples simultaneously for up to 4 generations. These easy-to-use devices can be provided in kit format, and will accelerate discoveries in diverse fields ranging from microbiology to systems and synthetic biology.

Evaluating the toxicity of bDtBPP on CHO-K1 cells for testing of single-use bioprocessing systems considering media selection, cell culture volume, mixing, and exposure duration.

PubMed

Shah, Rhythm R; Linville, Taylor W; Whynot, Andrew D; Brazel, Christopher S

2016-09-01

Single-use bioprocessing bags are gaining popularity due to ease of use, lower risk of contamination, and ease of process scale-up. Bis(2,4-di-tert-butylphenyl)phosphate (bDtBPP), a degradant of tris(2,4-di-tert-butylphenyl)phosphite, marketed as Irgafos 168®, which is an antioxidant stabilizer added to resins, has been identified as a potentially toxic leachate which may impact the performance of single-use, multilayer bioprocessing bags. In this study, the toxicity of bDtBPP was tested on CHO-K1 cells grown as adherent or suspended cells. The EC50 (effective concentration to cause 50% cell death) for adherent cells was found to be one order of magnitude higher than that for suspended CHO-K1 cells. While CHO-K1 cells had good cell viability when exposed to moderate concentrations of bDtBPP, the degradant was shown to impact the viable cell density (VCD) at much lower concentrations. Hence, in developing an industry-standard assay for testing the cytotoxicity of leachates, suspended cells (as commonly used in the bioprocessing industry) would likely be most sensitive, particularly when reporting EC50 values based on VCD. The effects of mixing, cell culture volume, and exposure duration were also evaluated for suspended CHO-K1 cells. It was found that the sensitivity of cell culture to leachates from single-use plastic bags was enhanced for suspended cells cultured for longer exposure times and when the cells were subjected to continuous agitation, both of which are important considerations in the production of biopharmaceuticals. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1318-1323, 2016. © 2016 American Institute of Chemical Engineers.

Continuous bioluminescent monitoring of cytoplasmic ATP in single isolated rat hepatocytes during metabolic poisoning.

PubMed Central

Koop, A; Cobbold, P H

1993-01-01

We have devised a technique for monitoring cytoplasmic ATP continuously in single hepatocytes. Single isolated rat hepatocytes were injected with the ATP-dependent luminescent protein firefly luciferase, and then superfused with 45 microM luciferin in air-equilibrated medium. Signals of approx. 10-200 photoelectron counts per second could be recorded from individual healthy cells for up to 3 h. The response of the luminescent signal to chemical hypoxia (2-5 mM CN- and 5-10 mM 2-deoxyglucose) was monitored. We found a great cell-to-cell variability in the time course of the ATP decline in response to CN-, 2-deoxyglucose or to their combination; the time for the signal to fall to 10% of the original (corresponding to approx. 100 microM ATP) ranged from approx. 20 to 75 min. This resistance of the cytoplasmic ATP concentration to depletion after blockade of oxidative phosphorylation and glycolysis could be abolished by pretreatment of the cells with etomoxir, which blocks mitochondrial beta-oxidation. Etomoxir alone had no effect on the luciferase signal, but etomoxir-pre-treated cells showed a prompt fall in the luciferase signal starting within 1-2 min of application of cyanide and 2-deoxyglucose and falling to 10% of the original signal in approx. 6-10 min. The technique allows cytoplasmic ATP changes to be monitored in single hepatocytes at concentrations of 1 mM or lower, but more precise calibration of the signal will require correction for the effects of cytoplasmic pH changes. PMID:8216212

Expression of single-chain Fv gene specific for gamma-seminoprotein by RTS and its biological activity identification.

PubMed

Han, Yuedong; Haun, Yi; Deng, Jinlan; Gao, Feng; Pan, Bifeng; Cui, Daxiang

2006-01-01

Fabricating a single-chain variable fragment specific for human seminoprotein is very important in antibody-directed enzyme prodrug therapy and NMR imaging for prostate cancer. Here a single-chain Fv specific for gamma-seminoprotein was expressed by RTS. Its activity and the efficiency of entry into prostate cancer cells are investigated by immunoprecipitation and Western blotting and immunofluorescent staining, as well as entry of conjugated magnetic beads into cells. Results showed that ScFv peptides specific for gamma-seminoprotein were successfully prepared, which can bind with the prostate cells specifically and can bring magnetic beads into prostate cancer cells within 15 min, the amount of magnetic beads inside prostate cancer cells increased as the culture time prolonged. ScFv-conjugated magnetic beads did not enter into control cells. In conclusion, the ScFv peptide against human gamma-seminoprotein with biological activity was successfully fabricated, which can take magnetic beads to prostate cancer cells specifically and not to the control cells. This ScFv peptide against human gamma-seminoprotein should be useful in improving the detection and therapy of prostate cancer at early stages and NMR imaging.

Single organelle dynamics linked to 3D structure by correlative live-cell imaging and 3D electron microscopy.

PubMed

Fermie, Job; Liv, Nalan; Ten Brink, Corlinda; van Donselaar, Elly G; Müller, Wally H; Schieber, Nicole L; Schwab, Yannick; Gerritsen, Hans C; Klumperman, Judith

2018-05-01

Live-cell correlative light-electron microscopy (live-cell-CLEM) integrates live movies with the corresponding electron microscopy (EM) image, but a major challenge is to relate the dynamic characteristics of single organelles to their 3-dimensional (3D) ultrastructure. Here, we introduce focused ion beam scanning electron microscopy (FIB-SEM) in a modular live-cell-CLEM pipeline for a single organelle CLEM. We transfected cells with lysosomal-associated membrane protein 1-green fluorescent protein (LAMP-1-GFP), analyzed the dynamics of individual GFP-positive spots, and correlated these to their corresponding fine-architecture and immediate cellular environment. By FIB-SEM we quantitatively assessed morphological characteristics, like number of intraluminal vesicles and contact sites with endoplasmic reticulum and mitochondria. Hence, we present a novel way to integrate multiple parameters of subcellular dynamics and architecture onto a single organelle, which is relevant to address biological questions related to membrane trafficking, organelle biogenesis and positioning. Furthermore, by using CLEM to select regions of interest, our method allows for targeted FIB-SEM, which significantly reduces time required for image acquisition and data processing. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Co-infections of infectious spleen and kidney necrosis virus and Siniperca chuatsi rhabdovirus in Chinese perch (Siniperca chuatsi).

PubMed

Lin, Qiang; Fu, Xiaozhe; Li, Ningqiu; Wan, Quanyuan; Chen, Wenjie; Huang, Yunmao; Huang, Zhibin; Li, Jun; Zhao, Lijuan; Lin, Li

2017-10-01

In spite of the quite common co-infections of viruses in the cultured fish, most of the previous studies have just simply focused on the infection of a single pathogen. In this report, we observed that about 13% of cultured Chinese perch have been co-infected by infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV). Furthermore, Chinese perch could co-infected by ISKNV and SCRV by intraperitoneally injection with the two viruses. Interestingly, we revealed that the two viruses could even co-infect a single cell of Chinese perch in vivo and a single Chinese perch brain cells (CPB) cell in vitro. The dynamic co-infected viruses loads in the different tissues of Chinese perch showed dependent. When CPB cells were infected with the same 10 MOI of SCRV and ISKNV, the replication of SCRV overwhelmed the replication of ISKNV. When the MOI of ISKNV (10 MOI) was 10,000 times of MOI of SCRV (0.001 MOI), the dynamic virus loads of the two viruses in CPB cells indicated that co-infections could synergistically stimulate both viruses replication at the late time points but not at early time points. The co-infections of ISKNV and SCRV in the cultured Chinese perch will shed a new light on the prevention of the viral diseases of Chinese perch. The development of multivalent vaccine which could be effective for preventing against the co-infections of the viruses is highly needed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Design of quantum dot-conjugated lipids for long-term, high-speed tracking experiments on cell surfaces.

PubMed

Murcia, Michael J; Minner, Daniel E; Mustata, Gina-Mirela; Ritchie, Kenneth; Naumann, Christoph A

2008-11-12

The current study reports the facile design of quantum dot (QD)-conjugated lipids and their application to high-speed tracking experiments on cell surfaces. CdSe/ZnS core/shell QDs with two types of hydrophilic coatings, 2-(2-aminoethoxy)ethanol (AEE) and a 60:40 molar mixture of 1,2-dipalmitoyl- sn-glycero-3-phosphocholine and 1,2-dipalmitoyl- sn-glycero-3-phosphoethanolamine- N-[methoxy(polyethylene glycol-2000], are conjugated to sulfhydryl lipids via maleimide reactive groups on the QD surface. Prior to lipid conjugation, the colloidal stability of both types of coated QDs in aqueous solution is confirmed using fluorescence correlation spectroscopy. A sensitive assay based on single lipid tracking experiments on a planar solid-supported phospholipid bilayer is presented that establishes conditions of monovalent conjugation of QDs to lipids. The QD-lipids are then employed as single-molecule tracking probes in plasma membranes of several cell types. Initial tracking experiments at a frame rate of 30 frames/s corroborate that QD-lipids diffuse like dye-labeled lipids in the plasma membrane of COS-7, HEK-293, 3T3, and NRK cells, thus confirming monovalent labeling. Finally, QD-lipids are applied for the first time to high-speed single-molecule imaging by tracking their lateral mobility in the plasma membrane of NRK fibroblasts with up to 1000 frames/s. Our high-speed tracking data, which are in excellent agreement with previous tracking experiments that used larger (40 nm) Au labels, not only push the time resolution in long-time, continuous fluorescence-based single-molecule tracking but also show that highly photostable, photoluminescent nanoprobes of 10 nm size can be employed (AEE-coated QDs). These probes are also attractive because, unlike Au nanoparticles, they facilitate complex multicolor experiments.

Apoptosis of Lewis Lung Carcinoma Cells Induced by Microwave via p53 and Proapoptotic Proteins In vivo.

PubMed

Zhang, Kou-Dong; Tong, Lin-Rong; Wang, Shui-Ming; Peng, Rui-Yun; Huang, Hai-Dong; Dong, Yu-Chao; Zhang, Xing-Xing; Li, Qiang; Bai, Chong

Microwave therapy is a minimal invasive procedure and has been employed in clinical practice for the treatment of various types of cancers. However, its therapeutic application in non-small-cell lung cancer and the underlying mechanism remains to be investigated. This study aimed to investigate its effect on Lewis lung carcinoma (LLC) tumor in vivo. Fifty LLC tumor-bearing C57BL/6 mice were adopted to assess the effect of microwave radiation on the growth and apoptosis of LLC tumor in vivo. These mice were randomly assigned to 10 groups with 5 mice in each group. Five groups were treated by single pulse microwave at different doses for different time, and the other five groups were radiated by multiple-pulse treatment of a single dose. Apoptosis of cancer cells was determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Western blotting was applied to detect the expression of proteins. Single pulse of microwave radiation for 5 min had little effect on the mice. Only 15-min microwave radiation at 30 mW/cm2 significantly increased the mice body temperature (2.20 ± 0.82)°C as compared with the other groups (0.78 ± 0.29 °C, 1.24 ± 0.52 °C, 0.78 ± 0.42 °C, respectively), but it did not affect the apoptosis of LLC tumor cells significantly. Continous microwave radiation exposure, single dose microwave radiation once per day for up to seven days, inhibited cell division and induced apoptosis of LLC tumor cells in a dose- and duration-dependent manner. It upregulated the protein levels of p53, Caspase 3, Bax and downregulated Bcl-2 protein. Multiple exposures of LLC-bearing mice to microwave radiation effectively induced tumor cell apoptosis at least partly by upregulating proapoptotic proteins and downregulating antiapoptotic proteins. Continuous radiation at low microwave intensity for a short time per day is promising in treating non-small-cell lung cancer.

Feasibility of mesenchymal stem cell culture expansion for a phase I clinical trial in multiple sclerosis.

PubMed

Planchon, Sarah M; Lingas, Karen T; Reese Koç, Jane; Hooper, Brittney M; Maitra, Basabi; Fox, Robert M; Imrey, Peter B; Drake, Kylie M; Aldred, Micheala A; Lazarus, Hillard M; Cohen, Jeffrey A

2018-01-01

Multiple sclerosis is an inflammatory, neurodegenerative disease of the central nervous system for which therapeutic mesenchymal stem cell transplantation is under study. Published experience of culture-expanding multiple sclerosis patients' mesenchymal stem cells for clinical trials is limited. To determine the feasibility of culture-expanding multiple sclerosis patients' mesenchymal stem cells for clinical use. In a phase I trial, autologous, bone marrow-derived mesenchymal stem cells were isolated from 25 trial participants with multiple sclerosis and eight matched controls, and culture-expanded to a target single dose of 1-2 × 10 6 cells/kg. Viability, cell product identity and sterility were assessed prior to infusion. Cytogenetic stability was assessed by single nucleotide polymorphism analysis of mesenchymal stem cells from 18 multiple sclerosis patients and five controls. One patient failed screening. Mesenchymal stem cell culture expansion was successful for 24 of 25 multiple sclerosis patients and six of eight controls. The target dose was achieved in 16-62 days, requiring two to three cell passages. Growth rate and culture success did not correlate with demographic or multiple sclerosis disease characteristics. Cytogenetic studies identified changes on one chromosome of one control (4.3%) after extended time in culture. Culture expansion of mesenchymal stem cells from multiple sclerosis patients as donors is feasible. However, culture time should be minimized for cell products designated for therapeutic administration.

Visualization of tandem repeat mutagenesis in Bacillus subtilis.

PubMed

Dormeyer, Miriam; Lentes, Sabine; Ballin, Patrick; Wilkens, Markus; Klumpp, Stefan; Kohlheyer, Dietrich; Stannek, Lorena; Grünberger, Alexander; Commichau, Fabian M

2018-03-01

Mutations are crucial for the emergence and evolution of proteins with novel functions, and thus for the diversity of life. Tandem repeats (TRs) are mutational hot spots that are present in the genomes of all organisms. Understanding the molecular mechanism underlying TR mutagenesis at the level of single cells requires the development of mutation reporter systems. Here, we present a mutation reporter system that is suitable to visualize mutagenesis of TRs occurring in single cells of the Gram-positive model bacterium Bacillus subtilis using microfluidic single-cell cultivation. The system allows measuring the elimination of TR units due to growth rate recovery. The cultivation of bacteria carrying the mutation reporter system in microfluidic chambers allowed us for the first time to visualize the emergence of a specific mutation at the level of single cells. The application of the mutation reporter system in combination with microfluidics might be helpful to elucidate the molecular mechanism underlying TR (in)stability in bacteria. Moreover, the mutation reporter system might be useful to assess whether mutations occur in response to nutrient starvation. Copyright © 2018 Elsevier B.V. All rights reserved.

Free stream capturing in fluid conservation law for moving coordinates in three dimensions

NASA Technical Reports Server (NTRS)

Obayashi, Shigeru

1991-01-01

The free-stream capturing technique for both the finite-volume (FV) and finite-difference (FD) framework is summarized. For an arbitrary motion of the grid, the FV analysis shows that volumes swept by all six surfaces of the cell have to be computed correctly. This means that the free-stream capturing time-metric terms should be calculated not only from a surface vector of a cell at a single time level, but also from a volume swept by the cell surface in space and time. The FV free-stream capturing formulation is applicable to the FD formulation by proper translation from an FV cell to an FD mesh.

Multiple mutant clones in blood rarely coexist

NASA Astrophysics Data System (ADS)

Dingli, David; Pacheco, Jorge M.; Traulsen, Arne

2008-02-01

Leukemias arise due to mutations in the genome of hematopoietic (blood) cells. Hematopoiesis has a multicompartment architecture, with cells exhibiting different rates of replication and differentiation. At the root of this process, one finds a small number of stem cells, and hence the description of the mutation-selection dynamics of blood cells calls for a stochastic approach. We use stochastic dynamics to investigate to which extent acquired hematopoietic disorders are associated with mutations of single or multiple genes within developing blood cells. Our analysis considers the appearance of mutations both in the stem cell compartment as well as in more committed compartments. We conclude that in the absence of genomic instability, acquired hematopoietic disorders due to mutations in multiple genes are most likely very rare events, as multiple mutations typically require much longer development times compared to those associated with a single mutation.

Imaging of single liver tumor cells intoxicated by heavy metals using ToF-SIMS

NASA Astrophysics Data System (ADS)

Mai, Fu-Der; Chen, Bo-Jung; Wu, Li-Chen; Li, Feng-Yin; Chen, Wen-Kang

2006-07-01

Human liver tumor cells intoxicated with five different Cd, Cu, Cr, Hg and Zn metals were analyzed using imaging time-of-flight secondary ion mass spectrometry (ToF-SIMS) to visualize the metal distributions in a single cell basis. A protocol was developed by combining rapid freezing, freeze-fracture and imprinting for transferring the intoxicated cells to a silicon wafer. As shown in the ToF-SIMS images, the cellular morphology was preserved indicating that this protocol can be used to prepare a representative cell for ToF-SIMS imaging analysis. Among the five metal ions investigated in this study, only Cr and Cu ions show preferential diffusion into the cell after simulated intoxication while the signals of the other three ions are either too low to be detected or unable to be distinguished from background intensity.

Millimeter-wave irradiation heating for operation of doped CeO2 electrolyte-supported single solid oxide fuel cell

NASA Astrophysics Data System (ADS)

Che Abdullah, Salmie Suhana Binti; Teranishi, Takashi; Hayashi, Hidetaka; Kishimoto, Akira

2018-01-01

High operation temperature of solid oxide fuel cell (SOFC) results in high cell and operation cost, time consuming and fast cell degradation. Developing high performance SOFC that operates at lower temperature is required. Here we demonstrate 24 GHz microwave as a rapid heating source to replace conventional heating method for SOFC operation using 20 mol% Sm doped CeO2 electrolyte-supported single cell. The tested cell shows improvement of 62% in maximum power density at 630 °C under microwave heating. This improvement governs by bulk conductivity of the electrolyte. Investigation of ionic transference number reveals that the value is unchanged under microwave irradiation, confirming the charge carrier is dominated by oxygen ion species. This work shows a potential new concept of high performance as well as cost and energy effective SOFC.

Cell size control and homeostasis in bacteria

NASA Astrophysics Data System (ADS)

Bradde, Serena; Taheri, Sattar; Sauls, John; Hill, Nobert; Levine, Petra; Paulsson, Johan; Vergassola, Massimo; Jun, Suckjoon

2015-03-01

How cells control their size is a fundamental question in biology. The mechanisms for sensing size, time, or a combination of the two are not supported by experimental evidence. By analysing distributions of size at division at birth and generation time of hundreds of thousands of Gram-negative E. coli and Gram-positive B. subtilis cells under a wide range of tightly controlled steady-state growth conditions, we are now in the position to validate different theoretical models. In this talk I will present all possible models in details and present a general mechanism that quantitatively explains all measurable aspects of growth and cell division at both population and single-cell levels.

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM).

PubMed

Tang, Anson H L; Lai, Queenie T K; Chung, Bob M F; Lee, Kelvin C M; Mok, Aaron T Y; Yip, G K; Shum, Anderson H C; Wong, Kenneth K Y; Tsia, Kevin K

2017-06-28

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture - information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization backend), as well as the workflow of imaging flow cytometry based on ATOM, using human cells and micro-algae as the examples.

Analysis of long-time operation of micro-cogeneration unit with fuel cell

NASA Astrophysics Data System (ADS)

Patsch, Marek; Čaja, Alexander

2015-05-01

Micro-cogeneration is cogeneration with small performance, with maximal electric power up to 50 kWe. On the present, there are available small micro-cogeneration units with small electric performance, about 1 kWe, which are usable also in single family houses or flats. These micro-cogeneration units operate on principle of conventional combustion engine, Stirling engine, steam engine or fuel cell. Micro-cogeneration units with fuel cells are new progressive developing type of units for single family houses. Fuel cell is electrochemical device which by oxidation-reduction reaction turn directly chemical energy of fuel to electric power, secondary products are pure water and thermal energy. The aim of paper is measuring and evaluation of operation parameters of micro-cogeneration unit with fuel cell which uses natural gas as a fuel.

BK/TD models for analyzing in vitro impedance data on cytotoxicity.

PubMed

Teng, S; Barcellini-Couget, S; Beaudouin, R; Brochot, C; Desousa, G; Rahmani, R; Pery, A R R

2015-06-01

The ban of animal testing has enhanced the development of new in vitro technologies for cosmetics safety assessment. Impedance metrics is one such technology which enables monitoring of cell viability in real time. However, analyzing real time data requires moving from static to dynamic toxicity assessment. In the present study, we built mechanistic biokinetic/toxicodynamic (BK/TD) models to analyze the time course of cell viability in cytotoxicity assay using impedance. These models account for the fate of the tested compounds during the assay. BK/TD models were applied to analyze HepaRG cell viability, after single (48 h) and repeated (4 weeks) exposures to three hepatotoxic compounds (coumarin, isoeugenol and benzophenone-2). The BK/TD models properly fit the data used for their calibration that was obtained for single or repeated exposure. Only for one out of the three compounds, the models calibrated with a single exposure were able to predict repeated exposure data. We therefore recommend the use of long-term exposure in vitro data in order to adequately account for chronic hepatotoxic effects. The models we propose here are capable of being coupled with human biokinetic models in order to relate dose exposure and human hepatotoxicity. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Tracking Image Correlation: Combining Single-Particle Tracking and Image Correlation

PubMed Central

Dupont, A.; Stirnnagel, K.; Lindemann, D.; Lamb, D.C.

2013-01-01

The interactions and coordination of biomolecules are crucial for most cellular functions. The observation of protein interactions in live cells may provide a better understanding of the underlying mechanisms. After fluorescent labeling of the interacting partners and live-cell microscopy, the colocalization is generally analyzed by quantitative global methods. Recent studies have addressed questions regarding the individual colocalization of moving biomolecules, usually by using single-particle tracking (SPT) and comparing the fluorescent intensities in both color channels. Here, we introduce a new method that combines SPT and correlation methods to obtain a dynamical 3D colocalization analysis along single trajectories of dual-colored particles. After 3D tracking, the colocalization is computed at each particle’s position via the local 3D image cross correlation of the two detection channels. For every particle analyzed, the output consists of the 3D trajectory, the time-resolved 3D colocalization information, and the fluorescence intensity in both channels. In addition, the cross-correlation analysis shows the 3D relative movement of the two fluorescent labels with an accuracy of 30 nm. We apply this method to the tracking of viral fusion events in live cells and demonstrate its capacity to obtain the time-resolved colocalization status of single particles in dense and noisy environments. PMID:23746509

Characterization of the COD removal, electricity generation, and bacterial communities in microbial fuel cells treating molasses wastewater.

PubMed

Lee, Yun-Yeong; Kim, Tae G; Cho, Kyung-Suk

2016-11-09

The chemical oxygen demand (COD) removal, electricity generation, and microbial communities were compared in 3 types of microbial fuel cells (MFCs) treating molasses wastewater. Single-chamber MFCs without and with a proton exchange membrane (PEM), and double-chamber MFC were constructed. A total of 10,000 mg L(-1) COD of molasses wastewater was continuously fed. The COD removal, electricity generation, and microbial communities in the two types of single-chamber MFCs were similar, indicating that the PEM did not enhance the reactor performance. The COD removal in the single-chamber MFCs (89-90%) was higher than that in the double-chamber MFC (50%). However, electricity generation in the double-chamber MFC was higher than that in the single-chamber MFCs. The current density (80 mA m(-2)) and power density (17 mW m(-2)) in the double-chamber MFC were 1.4- and 2.2-times higher than those in the single-chamber MFCs, respectively. The bacterial community structures in single- and double-chamber MFCs were also distinguishable. The amount of Proteobacteria in the double-chamber MFC was 2-3 times higher than those in the single-chamber MFCs. For the archaeal community, Methanothrix (96.4%) was remarkably dominant in the single-chamber MFCs, but Methanobacterium (35.1%), Methanosarcina (28.3%), and Methanothrix (16.2%) were abundant in the double-chamber MFC.

Single-Cell Resolution of Temporal Gene Expression during Heart Development.

PubMed

DeLaughter, Daniel M; Bick, Alexander G; Wakimoto, Hiroko; McKean, David; Gorham, Joshua M; Kathiriya, Irfan S; Hinson, John T; Homsy, Jason; Gray, Jesse; Pu, William; Bruneau, Benoit G; Seidman, J G; Seidman, Christine E

2016-11-21

Activation of complex molecular programs in specific cell lineages governs mammalian heart development, from a primordial linear tube to a four-chamber organ. To characterize lineage-specific, spatiotemporal developmental programs, we performed single-cell RNA sequencing of >1,200 murine cells isolated at seven time points spanning embryonic day 9.5 (primordial heart tube) to postnatal day 21 (mature heart). Using unbiased transcriptional data, we classified cardiomyocytes, endothelial cells, and fibroblast-enriched cells, thus identifying markers for temporal and chamber-specific developmental programs. By harnessing these datasets, we defined developmental ages of human and mouse pluripotent stem-cell-derived cardiomyocytes and characterized lineage-specific maturation defects in hearts of mice with heterozygous mutations in Nkx2.5 that cause human heart malformations. This spatiotemporal transcriptome analysis of heart development reveals lineage-specific gene programs underlying normal cardiac development and congenital heart disease. Copyright © 2016 Elsevier Inc. All rights reserved.

Just-in-time vaccines: Biomineralized calcium phosphate core-immunogen shell nanoparticles induce long-lasting CD8+ T cell responses in mice

PubMed Central

Zhou, Weibin; Moguche, Albanus; Chiu, David; Murali-Krishna, Kaja; Baneyx, François

2014-01-01

Distributed and on-demand vaccine production could be game-changing for infectious disease treatment in the developing world by providing new therapeutic opportunities and breaking the refrigeration “cold chain”. Here, we show that a fusion protein between a calcium phosphate binding domain and the model antigen ovalbumin can mineralize a biocompatible adjuvant in a single step. The resulting 50 nm calcium phosphate core-immunogen shell particles are comparable to soluble protein in inducing ovalbumin-specific antibody response and class switch recombination in mice. However, single dose vaccination with nanoparticles leads to higher expansion of ovalbumin-specific CD8+ T cells upon challenge with an influenza virus bearing the ovalbumin-derived SIINFEKL peptide, and these cells produce high levels of IFN-γ. Furthermore, mice exhibit a robust antigen-specific CD8+ T cell recall response when challenged with virus 8 months post-immunization. These results underscore the promise of immunogen-controlled adjuvant mineralization for just-in-time manufacturing of effective T cell vaccines. PMID:24275478

High-performance imaging of stem cells using single-photon emissions

NASA Astrophysics Data System (ADS)

Wagenaar, Douglas J.; Moats, Rex A.; Hartsough, Neal E.; Meier, Dirk; Hugg, James W.; Yang, Tang; Gazit, Dan; Pelled, Gadi; Patt, Bradley E.

2011-10-01

Radiolabeled cells have been imaged for decades in the field of autoradiography. Recent advances in detector and microelectronics technologies have enabled the new field of "digital autoradiography" which remains limited to ex vivo specimens of thin tissue slices. The 3D field-of-view (FOV) of single cell imaging can be extended to millimeters if the low energy (10-30 keV) photon emissions of radionuclides are used for single-photon nuclear imaging. This new microscope uses a coded aperture foil made of highly attenuating elements such as gold or platinum to form the image as a kind of "lens". The detectors used for single-photon emission microscopy are typically silicon detectors with a pixel pitch less than 60 μm. The goal of this work is to image radiolabeled mesenchymal stem cells in vivo in an animal model of tendon repair processes. Single-photon nuclear imaging is an attractive modality for translational medicine since the labeled cells can be imaged simultaneously with the reparative processes by using the dual-isotope imaging technique. The details our microscope's two-layer gold aperture and the operation of the energy-dispersive, pixellated silicon detector are presented along with the first demonstration of energy discrimination with a 57Co source. Cell labeling techniques have been augmented by genetic engineering with the sodium-iodide symporter, a type of reporter gene imaging method that enables in vivo uptake of free 99mTc or an iodine isotope at a time point days or weeks after the insertion of the genetically modified stem cells into the animal model. This microscopy work in animal research may expand to the imaging of reporter-enabled stem cells simultaneously with the expected biological repair process in human clinical trials of stem cell therapies.

Determination of EGFR mutations in single cells microdissected from enriched lung tumor cells in peripheral blood.

PubMed

Ran, Ran; Li, Longyun; Wang, Mengzhao; Wang, Shulan; Zheng, Zhi; Lin, Peter Ping

2013-09-01

A minimally invasive and repeatable approach for real-time epidermal growth factor receptor (EGFR) mutation surveillance would be highly beneficial for individualized therapy of late stage lung cancer patients whose surgical specimens are often not available. We aim to develop a viable method to detect EGFR mutations in single circulating tumor cells (CTCs). Using a model CTC system of spiked tumor cells in whole blood, we evaluated EGFR mutation determination in single tumor cells enriched from blood. We used magnetic beads labeled with antibody against leukocyte surface antigens to deplete leukocytes and enrich native CTCs independent of epithelial marker expression level. We then used laser cell microdissection (LCM) to isolate individual CTCs, followed by whole-genome amplification of the DNA for exon 19 microdeletion, L858R and T790M mutation detection by PCR sequencing. EGFR mutations were successfully measured in individual spiked tumor cells enriched from 7.5 ml whole blood. Whole-genome amplification provided sufficient DNA for mutation determination at multiple sites. Ninety-five percent of the single CTCs microdissected by LCM (19/20) yielded PCR amplicons for at least one of the three mutation sites. The amplification success rates were 55 % (11/20) for exon 19 deletion, 45 % (9/20) for T790M, and 85 % (17/20) for L858R. Sequencing of the amplicons showed allele dropout in the amplification reactions, but mutations were correctly identified in 80 % of the amplicons. EGFR mutation determination from single captured tumor cells from blood is feasible with the approach described here. However, to overcome allele dropout and to obtain reliable information about the tumor's EGFR status, multiple individual tumor cells should be assayed.

Fundamental limits on dynamic inference from single-cell snapshots

PubMed Central

Weinreb, Caleb; Tusi, Betsabeh K.; Socolovsky, Merav

2018-01-01

Single-cell expression profiling reveals the molecular states of individual cells with unprecedented detail. Because these methods destroy cells in the process of analysis, they cannot measure how gene expression changes over time. However, some information on dynamics is present in the data: the continuum of molecular states in the population can reflect the trajectory of a typical cell. Many methods for extracting single-cell dynamics from population data have been proposed. However, all such attempts face a common limitation: for any measured distribution of cell states, there are multiple dynamics that could give rise to it, and by extension, multiple possibilities for underlying mechanisms of gene regulation. Here, we describe the aspects of gene expression dynamics that cannot be inferred from a static snapshot alone and identify assumptions necessary to constrain a unique solution for cell dynamics from static snapshots. We translate these constraints into a practical algorithmic approach, population balance analysis (PBA), which makes use of a method from spectral graph theory to solve a class of high-dimensional differential equations. We use simulations to show the strengths and limitations of PBA, and then apply it to single-cell profiles of hematopoietic progenitor cells (HPCs). Cell state predictions from this analysis agree with HPC fate assays reported in several papers over the past two decades. By highlighting the fundamental limits on dynamic inference faced by any method, our framework provides a rigorous basis for dynamic interpretation of a gene expression continuum and clarifies best experimental designs for trajectory reconstruction from static snapshot measurements. PMID:29463712

Effect of number and location of distant metastases on renal cell carcinoma mortality in candidates for cytoreductive nephrectomy: implications for multimodal therapy.

PubMed

Capitanio, Umberto; Abdollah, Firas; Matloob, Rayan; Salonia, Andrea; Suardi, Nazareno; Briganti, Alberto; Carenzi, Cristina; Rigatti, Patrizio; Montorsi, Francesco; Bertini, Roberto

2013-06-01

To test whether the combination of number and location of distant metastases affects cancer-specific survival in patients with metastatic renal cell carcinoma. Overall, 242 metastatic renal cell carcinoma patients with synchronous metastases at diagnosis underwent cytoreductive nephrectomy at a single institution. Combinations of number and location of distant metastases were coded as: single metastasis and single organ affected, multiple metastases and single organ affected, single metastasis for each of the multiple organs affected, and multiple metastases for each of the multiple organs affected. Covariates included age, symptoms, performance status, American Society of Anesthesiologists score, hemoglobin, lactate dehydrogenase, tumor size, Fuhrman grade, T stage, lymph node status, necrosis, sarcomatoid features and metastasectomy at the time of nephrectomy. The median survival was 34.7 versus 32.3 versus 29.6 versus 8.5 months for single metastasis and single organ affected, multiple metastases and single organ affected single metastasis for each of the multiple organs affected, and multiple metastases for each of the multiple organs affected patients, respectively. At multivariable analyses, the combination of number and location of distant metastases resulted in one of the most informative and independent predictors of cancer-specific survival in metastatic renal cell carcinoma patients. The lung was the location with the highest rate of single organ affected (50.3% vs 35.1% in other sites; P < 0.001). Considering only patients with a single metastasis, no statistically significantly different cancer-specific survival rates were recorded (P > 0.3) among different metastatic organs. Among metastatic renal cell carcinoma patients undergoing cytoreductive nephrectomy, the combination of the number and location of distant metastases is a major independent predictor of cancer-specific survival. Patients with multiple organs affected by multifocal disease are more likely to have poorer survival. © 2012 The Japanese Urological Association.

Multiphoton-generated localized electron plasma for membrane permeability modification in single cells

NASA Astrophysics Data System (ADS)

Merritt, T.; Leblanc, M.; McMillan, J.; Westwood, J.; Khodaparast, G. A.

2014-03-01

Successful incorporation of a specific macromolecule into a single cell would be ideal for characterizing trafficking dynamics through plasmodesmata or for studying intracellular localizations. Here, we demonstrate NIR femtosecond laser-mediated infiltration of a membrane impermeable dextran-conjugated dye into living cells of Arabidopsis thaliana seedling stems. Based on the reactions of fluorescing vacuoles of transgenic cells and artificial cell walls comprised of nanocellulose, laser intensity and exposure time were adjusted to avoid deleterious effects. Using these plant-tailored laser parameters, cells were injected with the fluorophores and long-term dye retention was observed, all while preserving vital cell functions. This method is ideal for studies concerning cell-to-cell interactions and potentially paves the way for introducing transgenes to specific cells. This work was supported by NSF award IOS-0843372 to JHW, with additional support from and U.S. Department of Agriculture Hatch Project no. 135997, and by the Institute of Critical Technology and Applied Sciences (ICTAS) at Virginia Tech.

Individuality and universality in the growth-division laws of single E. coli cells

NASA Astrophysics Data System (ADS)

Kennard, Andrew S.; Osella, Matteo; Javer, Avelino; Grilli, Jacopo; Nghe, Philippe; Tans, Sander J.; Cicuta, Pietro; Cosentino Lagomarsino, Marco

2016-01-01

The mean size of exponentially dividing Escherichia coli cells in different nutrient conditions is known to depend on the mean growth rate only. However, the joint fluctuations relating cell size, doubling time, and individual growth rate are only starting to be characterized. Recent studies in bacteria reported a universal trend where the spread in both size and doubling times is a linear function of the population means of these variables. Here we combine experiments and theory and use scaling concepts to elucidate the constraints posed by the second observation on the division control mechanism and on the joint fluctuations of sizes and doubling times. We found that scaling relations based on the means collapse both size and doubling-time distributions across different conditions and explain how the shape of their joint fluctuations deviates from the means. Our data on these joint fluctuations highlight the importance of cell individuality: Single cells do not follow the dependence observed for the means between size and either growth rate or inverse doubling time. Our calculations show that these results emerge from a broad class of division control mechanisms requiring a certain scaling form of the "division hazard rate function," which defines the probability rate of dividing as a function of measurable parameters. This "model free" approach gives a rationale for the universal body-size distributions observed in microbial ecosystems across many microbial species, presumably dividing with multiple mechanisms. Additionally, our experiments show a crossover between fast and slow growth in the relation between individual-cell growth rate and division time, which can be understood in terms of different regimes of genome replication control.

Quantitative analysis reveals crosstalk mechanisms of heat shock-induced attenuation of NF-κB signaling at the single cell level.

PubMed

Kardyńska, Małgorzata; Paszek, Anna; Śmieja, Jarosław; Spiller, David; Widłak, Wiesława; White, Michael R H; Paszek, Pawel; Kimmel, Marek

2018-04-01

Elevated temperature induces the heat shock (HS) response, which modulates cell proliferation, apoptosis, the immune and inflammatory responses. However, specific mechanisms linking the HS response pathways to major cellular signaling systems are not fully understood. Here we used integrated computational and experimental approaches to quantitatively analyze the crosstalk mechanisms between the HS-response and a master regulator of inflammation, cell proliferation, and apoptosis the Nuclear Factor κB (NF-κB) system. We found that populations of human osteosarcoma cells, exposed to a clinically relevant 43°C HS had an attenuated NF-κB p65 response to Tumor Necrosis Factor α (TNFα) treatment. The degree of inhibition of the NF-κB response depended on the HS exposure time. Mathematical modeling of single cells indicated that individual crosstalk mechanisms differentially encode HS-mediated NF-κB responses while being consistent with the observed population-level responses. In particular "all-or-nothing" encoding mechanisms were involved in the HS-dependent regulation of the IKK activity and IκBα phosphorylation, while others involving transport were "analogue". In order to discriminate between these mechanisms, we used live-cell imaging of nuclear translocations of the NF-κB p65 subunit. The single cell responses exhibited "all-or-nothing" encoding. While most cells did not respond to TNFα stimulation after a 60 min HS, 27% showed responses similar to those not receiving HS. We further demonstrated experimentally and theoretically that the predicted inhibition of IKK activity was consistent with the observed HS-dependent depletion of the IKKα and IKKβ subunits in whole cell lysates. However, a combination of "all-or-nothing" crosstalk mechanisms was required to completely recapitulate the single cell data. We postulate therefore that the heterogeneity of the single cell responses might be explained by the cell-intrinsic variability of HS-modulated IKK signaling. In summary, we show that high temperature modulates NF-κB responses in single cells in a complex and unintuitive manner, which needs to be considered in hyperthermia-based treatment strategies.

A software solution for recording circadian oscillator features in time-lapse live cell microscopy.

PubMed

Sage, Daniel; Unser, Michael; Salmon, Patrick; Dibner, Charna

2010-07-06

Fluorescent and bioluminescent time-lapse microscopy approaches have been successfully used to investigate molecular mechanisms underlying the mammalian circadian oscillator at the single cell level. However, most of the available software and common methods based on intensity-threshold segmentation and frame-to-frame tracking are not applicable in these experiments. This is due to cell movement and dramatic changes in the fluorescent/bioluminescent reporter protein during the circadian cycle, with the lowest expression level very close to the background intensity. At present, the standard approach to analyze data sets obtained from time lapse microscopy is either manual tracking or application of generic image-processing software/dedicated tracking software. To our knowledge, these existing software solutions for manual and automatic tracking have strong limitations in tracking individual cells if their plane shifts. In an attempt to improve existing methodology of time-lapse tracking of a large number of moving cells, we have developed a semi-automatic software package. It extracts the trajectory of the cells by tracking theirs displacements, makes the delineation of cell nucleus or whole cell, and finally yields measurements of various features, like reporter protein expression level or cell displacement. As an example, we present here single cell circadian pattern and motility analysis of NIH3T3 mouse fibroblasts expressing a fluorescent circadian reporter protein. Using Circadian Gene Express plugin, we performed fast and nonbiased analysis of large fluorescent time lapse microscopy datasets. Our software solution, Circadian Gene Express (CGE), is easy to use and allows precise and semi-automatic tracking of moving cells over longer period of time. In spite of significant circadian variations in protein expression with extremely low expression levels at the valley phase, CGE allows accurate and efficient recording of large number of cell parameters, including level of reporter protein expression, velocity, direction of movement, and others. CGE proves to be useful for the analysis of widefield fluorescent microscopy datasets, as well as for bioluminescence imaging. Moreover, it might be easily adaptable for confocal image analysis by manually choosing one of the focal planes of each z-stack of the various time points of a time series. CGE is a Java plugin for ImageJ; it is freely available at: http://bigwww.epfl.ch/sage/soft/circadian/.

Dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live cells

PubMed Central

McQuilken, Molly; La Riviere, Patrick J.; Occhipinti, Patricia; Verma, Amitabh; Oldenbourg, Rudolf; Gladfelter, Amy S.; Tani, Tomomi

2016-01-01

Regulation of order, such as orientation and conformation, drives the function of most molecular assemblies in living cells but remains difficult to measure accurately through space and time. We built an instantaneous fluorescence polarization microscope, which simultaneously images position and orientation of fluorophores in living cells with single-molecule sensitivity and a time resolution of 100 ms. We developed image acquisition and analysis methods to track single particles that interact with higher-order assemblies of molecules. We tracked the fluctuations in position and orientation of molecules from the level of an ensemble of fluorophores down to single fluorophores. We tested our system in vitro using fluorescently labeled DNA and F-actin, in which the ensemble orientation of polarized fluorescence is known. We then tracked the orientation of sparsely labeled F-actin network at the leading edge of migrating human keratinocytes, revealing the anisotropic distribution of actin filaments relative to the local retrograde flow of the F-actin network. Additionally, we analyzed the position and orientation of septin-GFP molecules incorporated in septin bundles in growing hyphae of a filamentous fungus. Our data indicate that septin-GFP molecules undergo positional fluctuations within ∼350 nm of the binding site and angular fluctuations within ∼30° of the central orientation of the bundle. By reporting position and orientation of molecules while they form dynamic higher-order structures, our approach can provide insights into how micrometer-scale ordered assemblies emerge from nanoscale molecules in living cells. PMID:27679846

Finite Element Analysis of Single Cell Stiffness Measurements Using PZT-Integrated Buckling Nanoneedles.

PubMed

Rad, Maryam Alsadat; Tijjani, Auwal Shehu; Ahmad, Mohd Ridzuan; Auwal, Shehu Muhammad

2016-12-23

This paper proposes a new technique for real-time single cell stiffness measurement using lead zirconate titanate (PZT)-integrated buckling nanoneedles. The PZT and the buckling part of the nanoneedle have been modelled and validated using the ABAQUS software. The two parts are integrated together to function as a single unit. After calibration, the stiffness, Young's modulus, Poisson's ratio and sensitivity of the PZT-integrated buckling nanoneedle have been determined to be 0.7100 N·m -1 , 123.4700 GPa, 0.3000 and 0.0693 V·m·N -1 , respectively. Three Saccharomyces cerevisiae cells have been modelled and validated based on compression tests. The average global stiffness and Young's modulus of the cells are determined to be 10.8867 ± 0.0094 N·m -1 and 110.7033 ± 0.0081 MPa, respectively. The nanoneedle and the cell have been assembled to measure the local stiffness of the single Saccharomyces cerevisiae cells The local stiffness, Young's modulus and PZT output voltage of the three different size Saccharomyces cerevisiae have been determined at different environmental conditions. We investigated that, at low temperature the stiffness value is low to adapt to the change in the environmental condition. As a result, Saccharomyces cerevisiae becomes vulnerable to viral and bacterial attacks. Therefore, the proposed technique will serve as a quick and accurate process to diagnose diseases at early stage in a cell for effective treatment.

The radiation hypersensitivity of cells at mitosis.

PubMed

Stobbe, C C; Park, S J; Chapman, J D

2002-12-01

Mitotic cells are hypersensitive to ionizing radiation, exhibiting single-hit inactivation coefficients near to those of repair deficient cell lines and lymphocytes. To elucidate possible mechanisms for this hypersensitivity, the kinetics of oxygen radiosensitization, the proportion of indirect effect by OH radicals and the kinetics of radiation-induced DNA strand breakage in the chromatin of mitotic cells were investigated. Synchronized populations of >90% mitotic HT-29 cells were obtained by the mitotic shake-off method. Cells were irradiated at < or =4 degrees C with (137)Cs gamma-rays. Cellular oxygen concentration was varied by gassing cell suspensions prior to and during irradiation with mixtures of pure N(2) that contained 5% CO(2) and measured quantities of O(2). The indirect effect of OH radicals was investigated with the radical scavenger, DMSO. DNA strand breakage was measured by the comet assay. Mitotic HT-29 cell inactivation is well described by a single-hit inactivation coefficient (alpha) of 1.14 +/- 0.06 Gy(-1). The oxygen enhancement ratio of mitotic cells (at 10% survival) was found to be approximately 2.0, significantly lower than the value of 2.8 measured for interphase (asynchronous) cells. More than 60% of mitotic cell killing was eliminated when the media contained 2 M DMSO, indicating that indirect effect is as important in the killing of mitotic cells as it is for interphase cells. The chromatin in mitotic cells was found to be ~2.8 times more sensitive to radiation-induced DNA single-strand breakage than the chromatin of interphase cells. The alpha-inactivation coefficient of mitotic HT-29 cells was ~30 times larger than that of interphase cells. Mitotic cell chromatin appears to contain intrinsic DNA breaks that are not lethal. In addition, chromatin in mitotic cells was found to be more susceptible to radiation-induced DNA strand-breakage than the dispersed chromatin of interphase cells. How the enhanced production of these simple DNA lesions (that are usually reparable) translates into the lethal (non-reparable) events associated with alpha-inactivation is not known. The compaction/dispersion status of DNA throughout the cell cycle appears to be an important factor for determining intrinsic cell radiosensitivity and might be manipulated for radiotherapeutic advantage.

Spike-timing dependent plasticity in primate corticospinal connections induced during free behavior

PubMed Central

Nishimura, Yukio; Perlmutter, Steve I.; Eaton, Ryan W.; Fetz, Eberhard E.

2014-01-01

Motor learning and functional recovery from brain damage involve changes in the strength of synaptic connections between neurons. Relevant in vivo evidence on the underlying cellular mechanisms remains limited and indirect. We found that the strength of neural connections between motor cortex and spinal cord in monkeys can be modified with an autonomous recurrent neural interface that delivers electrical stimuli in the spinal cord triggered by action potentials of corticospinal cells during free behavior. The activity-dependent stimulation modified the strength of the terminal connections of single corticomotoneuronal cells, consistent with a bidirectional spike-timing dependent plasticity rule previously derived from in vitro experiments. For some cells the changes lasted for days after the end of conditioning, but most effects eventually reverted to preconditioning levels. These results provide the first direct evidence of corticospinal synaptic plasticity in vivo at the level of single neurons induced by normal firing patterns during free behavior. PMID:24210907

Reflection-mode micro-spherical fiber-optic probes for in vitro real-time and single-cell level pH sensing.

PubMed

Yang, Qingbo; Wang, Hanzheng; Lan, Xinwei; Cheng, Baokai; Chen, Sisi; Shi, Honglan; Xiao, Hai; Ma, Yinfa

2015-02-01

pH sensing at the single-cell level without negatively affecting living cells is very important but still a remaining issue in the biomedical studies. A 70 μm reflection-mode fiber-optic micro-pH sensor was designed and fabricated by dip-coating thin layer of organically modified aerogel onto a tapered spherical probe head. A pH sensitive fluorescent dye 2', 7'-Bis (2-carbonylethyl)-5(6)-carboxyfluorescein (BCECF) was employed and covalently bonded within the aerogel networks. By tuning the alkoxide mixing ratio and adjusting hexamethyldisilazane (HMDS) priming procedure, the sensor can be optimized to have high stability and pH sensing ability. The in vitro real-time sensing capability was then demonstrated in a simple spectroscopic way, and showed linear measurement responses with a pH resolution up to an average of 0.049 pH unit within a narrow, but biological meaningful pH range of 6.12-7.81. Its novel characterizations of high spatial resolution, reflection mode operation, fast response and high stability, great linear response within biological meaningful pH range and high pH resolutions, make this novel pH probe a very cost-effective tool for chemical/biological sensing, especially within the single cell level research field.

The effect of single large dose hydrocortisone treatment on IgM and IgG antibody production, morphological distribution of antibody producing cells and immunological memory

PubMed Central

Petrányi, Gy.; Benczúr, M.; Alföldy, P.

1971-01-01

The effect of a single subcutaneous dose of hydrocortisone (730 mg/kg ∼ 21-day LD50) on the haemolysin response of mice to sheep erythrocyte antigen was examined. Hydrocortisone was administered once at times varying from 5 days before immunization with sheep erythrocytes to 9 days after antigen. Total suppression of the 7S haemolysin titre was brought about by treatment with single dose of 750 mg/kg in the period 5 days prior to antigen until 2 days after antigen; at the same time, the titre of 19S haemolysin exceeded the control 19S titre. Microplaque assay on the 5th day failed to confirm total suppression of 7S antibody synthesis, as 4 per cent of the splenic plaque-forming cells produced 7S. The same assay failed to verify the augmentation of 19S production on a cellular level, as the number of 19S plaque-forming was significantly decreased. Hydrocortisone could be shown to influence the morphology of the 19S antibody producing cells by increasing the percentage of mature cell types. A selective depressing activity by hydrocortisone on 7S memory was found. The theoretical implications of these findings are discussed. PMID:4934137

Reflection-mode micro-spherical fiber-optic probes for in vitro real-time and single-cell level pH sensing

PubMed Central

Yang, Qingbo; Wang, Hanzheng; Lan, Xinwei; Cheng, Baokai; Chen, Sisi; Shi, Honglan; Xiao, Hai; Ma, Yinfa

2014-01-01

pH sensing at the single-cell level without negatively affecting living cells is very important but still a remaining issue in the biomedical studies. A 70 μm reflection-mode fiber-optic micro-pH sensor was designed and fabricated by dip-coating thin layer of organically modified aerogel onto a tapered spherical probe head. A pH sensitive fluorescent dye 2′, 7′-Bis (2-carbonylethyl)-5(6)-carboxyfluorescein (BCECF) was employed and covalently bonded within the aerogel networks. By tuning the alkoxide mixing ratio and adjusting hexamethyldisilazane (HMDS) priming procedure, the sensor can be optimized to have high stability and pH sensing ability. The in vitro real-time sensing capability was then demonstrated in a simple spectroscopic way, and showed linear measurement responses with a pH resolution up to an average of 0.049 pH unit within a narrow, but biological meaningful pH range of 6.12–7.81. Its novel characterizations of high spatial resolution, reflection mode operation, fast response and high stability, great linear response within biological meaningful pH range and high pH resolutions, make this novel pH probe a very cost-effective tool for chemical/biological sensing, especially within the single cell level research field. PMID:25530670

Impaired coordination between signaling pathways is revealed in human colorectal cancer using single-cell mass cytometry of archival tissue blocks

PubMed Central

Simmons, Alan J.; Scurrah, Cherie’ R.; McKinley, Eliot T.; Herring, Charles A.; Irish, Jonathan M.; Washington, Mary K.; Coffey, Robert J.; Lau, Ken S.

2016-01-01

Cellular heterogeneity poses a significant challenge to understanding tissue level phenotypes and confounds conventional bulk analyses. To facilitate the analysis of signaling at the single-cell level in human tissues, we applied mass cytometry using CyTOF (Cytometry Time-of-Flight) to formalin-fixed paraffin-embedded (FFPE) normal and diseased intestinal specimens. We developed and validated a technique called FFPE-DISSECT (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue), a single-cell approach for characterizing native signaling states from embedded solid tissue samples. We applied FFPE-DISSECT coupled to mass cytometry and found differential signaling by tumor necrosis factor α (TNF-α) in intestinal enterocytes, goblet cells and enteroendocrine cells, implicating the role of the downstream RAS-RAF-MEK-ERK signaling pathway in dictating goblet cell identity. In addition, application of FFPE-DISSECT, mass cytometry, and data-driven computational analyses to human colon specimens confirmed reduced differentiation in colorectal cancer (CRC) compared to normal colon, and revealed quantitative increases in inter- and intra-tissue heterogeneity in CRC with regards to the modular regulation of signaling pathways. Specifically, modular co-regulation of the kinases P38 and ERK, the translation regulator 4EBP1, and the transcription factor CREB in the proliferative compartment of the normal colon was loss in CRC, as evidenced by their impaired coordination over samplings of single cells in tissue. Our data suggest that this single-cell approach, applied in conjunction with genomic annotation, such as microsatellite instability and mutations in KRAS and BRAF, allows rapid and detailed characterization of cellular heterogeneity from clinical repositories of embedded human tissues. FFPE-DISSECT coupled of mass cytometry can be used for deriving cellular landscapes from archived patient samples, beyond CRC, and as a high resolution tool for disease characterization and subtyping. PMID:27729552

Optofluidic time-stretch microscopy: recent advances

NASA Astrophysics Data System (ADS)

Lei, Cheng; Nitta, Nao; Ozeki, Yasuyuki; Goda, Keisuke

2018-06-01

Flow cytometry is an indispensable method for valuable applications in numerous fields such as immunology, pathology, pharmacology, molecular biology, and marine biology. Optofluidic time-stretch microscopy is superior to conventional flow cytometry methods for its capability to acquire high-quality images of single cells at a high-throughput exceeding 10,000 cells per second. This makes it possible to extract copious information from cellular images for accurate cell detection and analysis with the assistance of machine learning. Optofluidic time-stretch microscopy has proven its effectivity in various applications, including microalga-based biofuel production, evaluation of thrombotic disorders, as well as drug screening and discovery. In this review, we discuss the principles and recent advances of optofluidic time-stretch microscopy.

Optofluidic time-stretch microscopy: recent advances

NASA Astrophysics Data System (ADS)

Lei, Cheng; Nitta, Nao; Ozeki, Yasuyuki; Goda, Keisuke

2018-04-01

Flow cytometry is an indispensable method for valuable applications in numerous fields such as immunology, pathology, pharmacology, molecular biology, and marine biology. Optofluidic time-stretch microscopy is superior to conventional flow cytometry methods for its capability to acquire high-quality images of single cells at a high-throughput exceeding 10,000 cells per second. This makes it possible to extract copious information from cellular images for accurate cell detection and analysis with the assistance of machine learning. Optofluidic time-stretch microscopy has proven its effectivity in various applications, including microalga-based biofuel production, evaluation of thrombotic disorders, as well as drug screening and discovery. In this review, we discuss the principles and recent advances of optofluidic time-stretch microscopy.

Imaging Stem Cells Implanted in Infarcted Myocardium

PubMed Central

Zhou, Rong; Acton, Paul D.; Ferrari, Victor A.

2008-01-01

Stem cell–based cellular cardiomyoplasty represents a promising therapy for myocardial infarction. Noninvasive imaging techniques would allow the evaluation of survival, migration, and differentiation status of implanted stem cells in the same subject over time. This review describes methods for cell visualization using several corresponding noninvasive imaging modalities, including magnetic resonance imaging, positron emission tomography, single-photon emission computed tomography, and bioluminescent imaging. Reporter-based cell visualization is compared with direct cell labeling for short- and long-term cell tracking. PMID:17112999

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

PubMed Central

Burke, Russell T.; Orth, James D.

2016-01-01

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls. PMID:27213923

bigSCale: an analytical framework for big-scale single-cell data.

PubMed

Iacono, Giovanni; Mereu, Elisabetta; Guillaumet-Adkins, Amy; Corominas, Roser; Cuscó, Ivon; Rodríguez-Esteban, Gustavo; Gut, Marta; Pérez-Jurado, Luis Alberto; Gut, Ivo; Heyn, Holger

2018-06-01

Single-cell RNA sequencing (scRNA-seq) has significantly deepened our insights into complex tissues, with the latest techniques capable of processing tens of thousands of cells simultaneously. Analyzing increasing numbers of cells, however, generates extremely large data sets, extending processing time and challenging computing resources. Current scRNA-seq analysis tools are not designed to interrogate large data sets and often lack sensitivity to identify marker genes. With bigSCale, we provide a scalable analytical framework to analyze millions of cells, which addresses the challenges associated with large data sets. To handle the noise and sparsity of scRNA-seq data, bigSCale uses large sample sizes to estimate an accurate numerical model of noise. The framework further includes modules for differential expression analysis, cell clustering, and marker identification. A directed convolution strategy allows processing of extremely large data sets, while preserving transcript information from individual cells. We evaluated the performance of bigSCale using both a biological model of aberrant gene expression in patient-derived neuronal progenitor cells and simulated data sets, which underlines the speed and accuracy in differential expression analysis. To test its applicability for large data sets, we applied bigSCale to assess 1.3 million cells from the mouse developing forebrain. Its directed down-sampling strategy accumulates information from single cells into index cell transcriptomes, thereby defining cellular clusters with improved resolution. Accordingly, index cell clusters identified rare populations, such as reelin ( Reln )-positive Cajal-Retzius neurons, for which we report previously unrecognized heterogeneity associated with distinct differentiation stages, spatial organization, and cellular function. Together, bigSCale presents a solution to address future challenges of large single-cell data sets. © 2018 Iacono et al.; Published by Cold Spring Harbor Laboratory Press.

Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells.

PubMed

Subach, Fedor V; Patterson, George H; Renz, Malte; Lippincott-Schwartz, Jennifer; Verkhusha, Vladislav V

2010-05-12

Rapidly emerging techniques of super-resolution single-molecule microscopy of living cells rely on the continued development of genetically encoded photoactivatable fluorescent proteins. On the basis of monomeric TagRFP, we have developed a photoactivatable TagRFP protein that is initially dark but becomes red fluorescent after violet light irradiation. Compared to other monomeric dark-to-red photoactivatable proteins including PAmCherry, PATagRFP has substantially higher molecular brightness, better pH stability, substantially less sensitivity to blue light, and better photostability in both ensemble and single-molecule modes. Spectroscopic analysis suggests that PATagRFP photoactivation is a two-step photochemical process involving sequential one-photon absorbance by two distinct chromophore forms. True monomeric behavior, absence of green fluorescence, and single-molecule performance in live cells make PATagRFP an excellent protein tag for two-color imaging techniques, including conventional diffraction-limited photoactivation microscopy, super-resolution photoactivated localization microscopy (PALM), and single particle tracking PALM (sptPALM) of living cells. Two-color sptPALM imaging was demonstrated using several PATagRFP tagged transmembrane proteins together with PAGFP-tagged clathrin light chain. Analysis of the resulting sptPALM images revealed that single-molecule transmembrane proteins, which are internalized into a cell via endocytosis, colocalize in space and time with plasma membrane domains enriched in clathrin light-chain molecules.

Single-step reinitialization and extending algorithms for level-set based multi-phase flow simulations

NASA Astrophysics Data System (ADS)

Fu, Lin; Hu, Xiangyu Y.; Adams, Nikolaus A.

2017-12-01

We propose efficient single-step formulations for reinitialization and extending algorithms, which are critical components of level-set based interface-tracking methods. The level-set field is reinitialized with a single-step (non iterative) "forward tracing" algorithm. A minimum set of cells is defined that describes the interface, and reinitialization employs only data from these cells. Fluid states are extrapolated or extended across the interface by a single-step "backward tracing" algorithm. Both algorithms, which are motivated by analogy to ray-tracing, avoid multiple block-boundary data exchanges that are inevitable for iterative reinitialization and extending approaches within a parallel-computing environment. The single-step algorithms are combined with a multi-resolution conservative sharp-interface method and validated by a wide range of benchmark test cases. We demonstrate that the proposed reinitialization method achieves second-order accuracy in conserving the volume of each phase. The interface location is invariant to reapplication of the single-step reinitialization. Generally, we observe smaller absolute errors than for standard iterative reinitialization on the same grid. The computational efficiency is higher than for the standard and typical high-order iterative reinitialization methods. We observe a 2- to 6-times efficiency improvement over the standard method for serial execution. The proposed single-step extending algorithm, which is commonly employed for assigning data to ghost cells with ghost-fluid or conservative interface interaction methods, shows about 10-times efficiency improvement over the standard method while maintaining same accuracy. Despite their simplicity, the proposed algorithms offer an efficient and robust alternative to iterative reinitialization and extending methods for level-set based multi-phase simulations.

MiRAR-miRNA Activity Reporter for Living Cells.

PubMed

Turk, Matthew A; Chung, Christina Z; Manni, Emad; Zukowski, Stephanie A; Engineer, Anish; Badakhshi, Yasaman; Bi, Yumin; Heinemann, Ilka U

2018-06-19

microRNA (miRNA) activity and regulation are of increasing interest as new therapeutic targets. Traditional approaches to assess miRNA levels in cells rely on RNA sequencing or quantitative PCR. While useful, these approaches are based on RNA extraction and cannot be applied in real-time to observe miRNA activity with single-cell resolution. We developed a green fluorescence protein (GFP)-based reporter system that allows for a direct, real-time readout of changes in miRNA activity in live cells. The miRNA activity reporter (MiRAR) consists of GFP fused to a 3′ untranslated region containing specific miRNA binding sites, resulting in miRNA activity-dependent GFP expression. Using qPCR, we verified the inverse relationship of GFP fluorescence and miRNA levels. We demonstrated that this novel optogenetic reporter system quantifies cellular levels of the tumor suppressor miRNA let-7 in real-time in single Human embryonic kidney 293 (HEK 293) cells. Our data shows that the MiRAR can be applied to detect changes in miRNA levels upon disruption of miRNA degradation pathways. We further show that the reporter could be adapted to monitor another disease-relevant miRNA, miR-122. With trivial modifications, this approach could be applied across the miRNome for quantification of many specific miRNA in cell cultures, tissues, or transgenic animal models.

Heritable stress response dynamics revealed by single-cell genealogy

PubMed Central

2018-01-01

Cells often respond to environmental stimuli by activating specific transcription factors. Upon exposure to glucose limitation stress, it is known that yeast Saccharomyces cerevisiae cells dephosphorylate the general stress response factor Msn2, leading to its nuclear localization, which in turn activates the expression of many genes. However, the precise dynamics of Msn2 nucleocytoplasmic translocations and whether they are inherited over multiple generations in a stress-dependent manner are not well understood. Tracking Msn2 localization events in yeast lineages grown on a microfluidic chip, here we report how cells modulate the amplitude, duration, frequency, and dynamic pattern of the localization events in response to glucose limitation stress. Single yeast cells were found to modulate the amplitude and frequency of Msn2 nuclear localization, but not its duration. Moreover, the Msn2 localization frequency was epigenetically inherited in descendants of mother cells, leading to a decrease in cell-to-cell variation in localization frequency. An analysis of the time dynamic patterns of nuclear localizations between genealogically related cell pairs using an information theory approach found that the magnitude of pattern similarity increased with stress intensity and was strongly inherited by the descendant cells at the highest stress level. By dissecting how general stress response dynamics is contributed by different modulation schemes over long time scales, our work provides insight into which scheme evolution might have acted on to optimize fitness in stressful environments. PMID:29675464

Visualization and quantitative analysis of extrachromosomal telomere-repeat DNA in individual human cells by Halo-FISH

PubMed Central

Komosa, Martin; Root, Heather; Meyn, M. Stephen

2015-01-01

Current methods for characterizing extrachromosomal nuclear DNA in mammalian cells do not permit single-cell analysis, are often semi-quantitative and frequently biased toward the detection of circular species. To overcome these limitations, we developed Halo-FISH to visualize and quantitatively analyze extrachromosomal DNA in single cells. We demonstrate Halo-FISH by using it to analyze extrachromosomal telomere-repeat (ECTR) in human cells that use the Alternative Lengthening of Telomeres (ALT) pathway(s) to maintain telomere lengths. We find that GM847 and VA13 ALT cells average ∼80 detectable G/C-strand ECTR DNA molecules/nucleus, while U2OS ALT cells average ∼18 molecules/nucleus. In comparison, human primary and telomerase-positive cells contain <5 ECTR DNA molecules/nucleus. ECTR DNA in ALT cells exhibit striking cell-to-cell variations in number (<20 to >300), range widely in length (<1 to >200 kb) and are composed of primarily G- or C-strand telomere-repeat DNA. Halo-FISH enables, for the first time, the simultaneous analysis of ECTR DNA and chromosomal telomeres in a single cell. We find that ECTR DNA comprises ∼15% of telomere-repeat DNA in GM847 and VA13 cells, but <4% in U2OS cells. In addition to its use in ALT cell analysis, Halo-FISH can facilitate the study of a wide variety of extrachromosomal DNA in mammalian cells. PMID:25662602

Intercellular signaling through secreted proteins induces free-energy gradient-directed cell movement.

PubMed

Kravchenko-Balasha, Nataly; Shin, Young Shik; Sutherland, Alex; Levine, R D; Heath, James R

2016-05-17

Controlling cell migration is important in tissue engineering and medicine. Cell motility depends on factors such as nutrient concentration gradients and soluble factor signaling. In particular, cell-cell signaling can depend on cell-cell separation distance and can influence cellular arrangements in bulk cultures. Here, we seek a physical-based approach, which identifies a potential governed by cell-cell signaling that induces a directed cell-cell motion. A single-cell barcode chip (SCBC) was used to experimentally interrogate secreted proteins in hundreds of isolated glioblastoma brain cancer cell pairs and to monitor their relative motions over time. We used these trajectories to identify a range of cell-cell separation distances where the signaling was most stable. We then used a thermodynamics-motivated analysis of secreted protein levels to characterize free-energy changes for different cell-cell distances. We show that glioblastoma cell-cell movement can be described as Brownian motion biased by cell-cell potential. To demonstrate that the free-energy potential as determined by the signaling is the driver of motion, we inhibited two proteins most involved in maintaining the free-energy gradient. Following inhibition, cell pairs showed an essentially random Brownian motion, similar to the case for untreated, isolated single cells.

Modeling cell-substrate de-adhesion dynamics under fluid shear

NASA Astrophysics Data System (ADS)

Maan, Renu; Rani, Garima; Menon, Gautam I.; Pullarkat, Pramod A.

2018-07-01

Changes in cell-substrate adhesion are believed to signal the onset of cancer metastasis, but such changes must be quantified against background levels of intrinsic heterogeneity between cells. Variations in cell-substrate adhesion strengths can be probed through biophysical measurements of cell detachment from substrates upon the application of an external force. Here, we investigate, theoretically and experimentally, the detachment of cells adhered to substrates when these cells are subjected to fluid shear. We present a theoretical framework within which we calculate the fraction of detached cells as a function of shear stress for fast ramps as well as the decay in this fraction at fixed shear stress as a function of time. Using HEK and 3T3 fibroblast cells as experimental model systems, we extract characteristic force scales for cell adhesion as well as characteristic detachment times. We estimate force-scales of  ∼500 pN associated to a single focal contact, and characteristic time-scales of s representing cell-spread-area dependent mean first passage times to the detached state at intermediate values of the shear stress. Variations in adhesion across cell types are especially prominent when cell detachment is probed by applying a time-varying shear stress. These methods can be applied to characterizing changes in cell adhesion in a variety of contexts, including metastasis.

Distributional behavior of diffusion coefficients obtained by single trajectories in annealed transit time model

NASA Astrophysics Data System (ADS)

Akimoto, Takuma; Yamamoto, Eiji

2016-12-01

Local diffusion coefficients in disordered systems such as spin glass systems and living cells are highly heterogeneous and may change over time. Such a time-dependent and spatially heterogeneous environment results in irreproducibility of single-particle-tracking measurements. Irreproducibility of time-averaged observables has been theoretically studied in the context of weak ergodicity breaking in stochastic processes. Here, we provide rigorous descriptions of equilibrium and non-equilibrium diffusion processes for the annealed transit time model, which is a heterogeneous diffusion model in living cells. We give analytical solutions for the mean square displacement (MSD) and the relative standard deviation of the time-averaged MSD for equilibrium and non-equilibrium situations. We find that the time-averaged MSD grows linearly with time and that the time-averaged diffusion coefficients are intrinsically random (irreproducible) even in the long-time measurements in non-equilibrium situations. Furthermore, the distribution of the time-averaged diffusion coefficients converges to a universal distribution in the sense that it does not depend on initial conditions. Our findings pave the way for a theoretical understanding of distributional behavior of the time-averaged diffusion coefficients in disordered systems.

A single frame: imaging live cells twenty-five years ago.

PubMed

Fink, Rachel

2011-07-01

In the mid-1980s live-cell imaging was changed by the introduction of video techniques, allowing new ways to collect and store data. The increased resolution obtained by manipulating video signals, the ability to use time-lapse videocassette recorders to study events that happen over long time intervals, and the introduction of fluorescent probes and sensitive video cameras opened research avenues previously unavailable. The author gives a personal account of this evolution, focusing on cell migration studies at the Marine Biological Laboratory 25 years ago. Copyright © 2011 Wiley-Liss, Inc.

Potentials of single-cell biology in identification and validation of disease biomarkers.

PubMed

Niu, Furong; Wang, Diane C; Lu, Jiapei; Wu, Wei; Wang, Xiangdong

2016-09-01

Single-cell biology is considered a new approach to identify and validate disease-specific biomarkers. However, the concern raised by clinicians is how to apply single-cell measurements for clinical practice, translate the message of single-cell systems biology into clinical phenotype or explain alterations of single-cell gene sequencing and function in patient response to therapies. This study is to address the importance and necessity of single-cell gene sequencing in the identification and development of disease-specific biomarkers, the definition and significance of single-cell biology and single-cell systems biology in the understanding of single-cell full picture, the development and establishment of whole-cell models in the validation of targeted biological function and the figure and meaning of single-molecule imaging in single cell to trace intra-single-cell molecule expression, signal, interaction and location. We headline the important role of single-cell biology in the discovery and development of disease-specific biomarkers with a special emphasis on understanding single-cell biological functions, e.g. mechanical phenotypes, single-cell biology, heterogeneity and organization of genome function. We have reason to believe that such multi-dimensional, multi-layer, multi-crossing and stereoscopic single-cell biology definitely benefits the discovery and development of disease-specific biomarkers. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Activation by intracellular GDP, metabolic inhibition and pinacidil of a glibenclamide-sensitive K-channel in smooth muscle cells of rat mesenteric artery.

PubMed Central

Zhang, H; Bolton, T B

1995-01-01

1. Single-channel recordings were made from cell-attached and isolated patches, and whole-cell currents were recorded under voltage clamp from single smooth muscle cells obtained by enzymic digestion of a small branch of the rat mesenteric artery. 2. In single voltage-clamped cells 1 mM uridine diphosphate (UDP) or guanidine diphosphate (GDP) added to the pipette solution, or pinacidil (100 microM) a K-channel opener (KCO) applied in the bathing solution, evoked an outward current of up to 100pA which was blocked by glibenclamide (10 microM). In single cells from which recordings were made by the 'perforated patch' (nystatin pipette) technique, metabolic inhibition by 1 mM NaCN and 10 mM 2-deoxy-glucose also evoked a similar glibenclamide-sensitive current. 3. Single K-channel activity was observed in cell-attached patches only infrequently unless the metabolism of the cell was inhibited, whereupon channel activity blocked by glibenclamide was seen; pinacidil applied to the cell evoked similar glibenclamide-sensitive channel activity. If the patch was pulled off the cell to form an isolated inside-out patch, similar glibenclamide-sensitive single-channel currents were observed in the presence of UDP and/or pinacidil to those seen in cell-attached mode; channel conductance was 20 pS (60:130 K-gradient) and openings showed no voltage-dependence and noisy inward currents, typical of the nucleoside diphosphate (NDP) activated K-channel (KNDP) seen previously in rabbit portal vein. 4. Formation of an isolated inside-out patch into an ATP-free solution did not increase the probability of channel opening which declined with time even when some single-channel activity had occurred in the cell-attached mode before detachment. However, application of 1 mM UDP or GDP, but not ATP, to inside-out patches evoked single-channel activity. Application of ATP-free solution to isolated patches, previously exposed to ATP and in which channel activity had been seen, did not evoke channel activity. 5. It is concluded that small conductance K-channels (KNDP) open in smooth muscle cells from this small artery in response to UDP or GDP acting from the inside, or pinacidil acting from the outside; the same channels open during inhibition of metabolism presumably mainly due to the rise in nucleoside diphosphates, but a fall in the ATP concentration on the inside of the channel did not by itself evoke channel activity.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7735693

Next-generation Sequencing (NGS) Analysis on Single Circulating Tumor Cells (CTCs) with No Need of Whole-genome Amplification (WGA).

PubMed

Palmirotta, Raffaele; Lovero, Domenica; Silvestris, Erica; Felici, Claudia; Quaresmini, Davide; Cafforio, Paola; Silvestris, Franco

2017-01-01

Isolation and genotyping of circulating tumor cells (CTCs) is gaining an increasing interest by clinical researchers in oncology not only for investigative purposes, but also for concrete application in clinical practice in terms of diagnosis, prognosis and decision treatment with targeted therapies. For the mutational analysis of single CTCs, the most advanced biotechnology methodology currently available includes the combination of whole genome amplification (WGA) followed by next-generation sequencing (NGS). However, the sequence of these molecular techniques is time-consuming and may also favor operator-dependent errors, related to the procedures themselves that, as in the case of the WGA technique, might affect downstream molecular analyses. A preliminary approach of molecular analysis by NGS on a model of CTCs without previous WGA procedural step was performed. We set-up an artificial sample obtained by spiking the SK-MEL-28 melanoma cell line in normal donor peripheral whole blood. Melanoma cells were first enriched using an AutoMACS® (Miltenyi) cell separator and then isolated as single and pooled CTCs by DEPArray™ System (Silicon Biosystems). NGS analysis, using the Ion AmpliSeq™ Cancer Hotspot Panel v2 (Life Technologies) with the Ion Torrent PGM™ system (Life Technologies), was performed on the SK-MEL-28 cell pellet, a single CTC previously processed with WGA and on 1, 2, 4 and 8 recovered CTCs without WGA pre-amplification. NGS directly carried out on CTCs without WGA showed the same mutations identified in SK-MEL-28 cell line pellet, with a considerable efficiency and avoiding the errors induced by the WGA procedure. We identified a cost-effective, time-saving and reliable methodological approach that could improve the analytical accuracy of the liquid biopsy and appears promising in studying CTCs from cancer patients for both research and clinical purposes. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Intravital imaging of cardiac function at the single-cell level.

PubMed

Aguirre, Aaron D; Vinegoni, Claudio; Sebas, Matt; Weissleder, Ralph

2014-08-05

Knowledge of cardiomyocyte biology is limited by the lack of methods to interrogate single-cell physiology in vivo. Here we show that contracting myocytes can indeed be imaged with optical microscopy at high temporal and spatial resolution in the beating murine heart, allowing visualization of individual sarcomeres and measurement of the single cardiomyocyte contractile cycle. Collectively, this has been enabled by efficient tissue stabilization, a prospective real-time cardiac gating approach, an image processing algorithm for motion-artifact-free imaging throughout the cardiac cycle, and a fluorescent membrane staining protocol. Quantification of cardiomyocyte contractile function in vivo opens many possibilities for investigating myocardial disease and therapeutic intervention at the cellular level.

Three dimensional time-gated tracking of non-blinking quantum dots in live cells

DOE PAGES

DeVore, Matthew S.; Werner, James H.; Goodwin, Peter M.; ...

2015-03-12

Single particle tracking has provided a wealth of information about biophysical processes such as motor protein transport and diffusion in cell membranes. However, motion out of the plane of the microscope or blinking of the fluorescent probe used as a label generally limits observation times to several seconds. Here, we overcome these limitations by using novel non-blinking quantum dots as probes and employing a custom 3D tracking microscope to actively follow motion in three dimensions (3D) in live cells. As a result, signal-to-noise is improved in the cellular milieu through the use of pulsed excitation and time-gated detection.

Pseudotemporal Ordering of Single Cells Reveals Metabolic Control of Postnatal β Cell Proliferation.

PubMed

Zeng, Chun; Mulas, Francesca; Sui, Yinghui; Guan, Tiffany; Miller, Nathanael; Tan, Yuliang; Liu, Fenfen; Jin, Wen; Carrano, Andrea C; Huising, Mark O; Shirihai, Orian S; Yeo, Gene W; Sander, Maike

2017-05-02

Pancreatic β cell mass for appropriate blood glucose control is established during early postnatal life. β cell proliferative capacity declines postnatally, but the extrinsic cues and intracellular signals that cause this decline remain unknown. To obtain a high-resolution map of β cell transcriptome dynamics after birth, we generated single-cell RNA-seq data of β cells from multiple postnatal time points and ordered cells based on transcriptional similarity using a new analytical tool. This analysis captured signatures of immature, proliferative β cells and established high expression of amino acid metabolic, mitochondrial, and Srf/Jun/Fos transcription factor genes as their hallmark feature. Experimental validation revealed high metabolic activity in immature β cells and a role for reactive oxygen species and Srf/Jun/Fos transcription factors in driving postnatal β cell proliferation and mass expansion. Our work provides the first high-resolution molecular characterization of state changes in postnatal β cells and paves the way for the identification of novel therapeutic targets to stimulate β cell regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

Suppression of lethal autoimmunity by regulatory T cells with a single TCR specificity

PubMed Central

Hemmers, Saskia; Schizas, Michail; Faire, Mehlika B.; Konopacki, Catherine; Schmidt-Supprian, Marc; Germain, Ronald N.

2017-01-01

The regulatory T cell (T reg cell) T cell receptor (TCR) repertoire is highly diverse and skewed toward recognition of self-antigens. TCR expression by T reg cells is continuously required for maintenance of immune tolerance and for a major part of their characteristic gene expression signature; however, it remains unknown to what degree diverse TCR-mediated interactions with cognate self-antigens are required for these processes. In this study, by experimentally switching the T reg cell TCR repertoire to a single T reg cell TCR, we demonstrate that T reg cell function and gene expression can be partially uncoupled from TCR diversity. An induced switch of the T reg cell TCR repertoire to a random repertoire also preserved, albeit to a limited degree, the ability to suppress lymphadenopathy and T helper cell type 2 activation. At the same time, these perturbations of the T reg cell TCR repertoire led to marked immune cell activation, tissue inflammation, and an ultimately severe autoimmunity, indicating the importance of diversity and specificity for optimal T reg cell function. PMID:28130403

Mass cytometry: a highly multiplexed single-cell technology for advancing drug development.

PubMed

Atkuri, Kondala R; Stevens, Jeffrey C; Neubert, Hendrik

2015-02-01

Advanced single-cell analysis technologies (e.g., mass cytometry) that help in multiplexing cellular measurements in limited-volume primary samples are critical in bridging discovery efforts to successful drug approval. Mass cytometry is the state-of-the-art technology in multiparametric single-cell analysis. Mass cytometers (also known as cytometry by time-of-flight or CyTOF) combine the cellular analysis principles of traditional fluorescence-based flow cytometry with the selectivity and quantitative power of inductively coupled plasma-mass spectrometry. Standard flow cytometry is limited in the number of parameters that can be measured owing to the overlap in signal when detecting fluorescently labeled antibodies. Mass cytometry uses antibodies tagged to stable isotopes of rare earth metals, which requires minimal signal compensation between the different metal tags. This unique feature enables researchers to seamlessly multiplex up to 40 independent measurements on single cells. In this overview we first present an overview of mass cytometry and compare it with traditional flow cytometry. We then discuss the emerging and potential applications of CyTOF technology in the pharmaceutical industry, including quantitative and qualitative deep profiling of immune cells and their applications in assessing drug immunogenicity, extensive mapping of signaling networks in single cells, cell surface receptor quantification and multiplexed internalization kinetics, multiplexing sample analysis by barcoding, and establishing cell ontologies on the basis of phenotype and/or function. We end with a discussion of the anticipated impact of this technology on drug development lifecycle with special emphasis on the utility of mass cytometry in deciphering a drug's pharmacokinetics and pharmacodynamics relationship. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Droplet-based microfluidic analysis and screening of single plant cells.

PubMed

Yu, Ziyi; Boehm, Christian R; Hibberd, Julian M; Abell, Chris; Haseloff, Jim; Burgess, Steven J; Reyna-Llorens, Ivan

2018-01-01

Droplet-based microfluidics has been used to facilitate high-throughput analysis of individual prokaryote and mammalian cells. However, there is a scarcity of similar workflows applicable to rapid phenotyping of plant systems where phenotyping analyses typically are time-consuming and low-throughput. We report on-chip encapsulation and analysis of protoplasts isolated from the emergent plant model Marchantia polymorpha at processing rates of >100,000 cells per hour. We use our microfluidic system to quantify the stochastic properties of a heat-inducible promoter across a population of transgenic protoplasts to demonstrate its potential for assessing gene expression activity in response to environmental conditions. We further demonstrate on-chip sorting of droplets containing YFP-expressing protoplasts from wild type cells using dielectrophoresis force. This work opens the door to droplet-based microfluidic analysis of plant cells for applications ranging from high-throughput characterisation of DNA parts to single-cell genomics to selection of rare plant phenotypes.

‘Particle genetics’: treating every cell as unique

PubMed Central

Yvert, Gaël

2014-01-01

Genotype-phenotype relations are usually inferred from a deterministic point of view. For example, quantitative trait loci (QTL), which describe regions of the genome associated with a particular phenotype, are based on a mean trait difference between genotype categories. However, living systems comprise huge numbers of cells (the ‘particles’ of biology). Each cell can exhibit substantial phenotypic individuality, which can have dramatic consequences at the organismal level. Now, with technology capable of interrogating individual cells, it is time to consider how genotypes shape the probability laws of single cell traits. The possibility of mapping single cell probabilistic trait loci (PTL), which link genomic regions to probabilities of cellular traits, is a promising step in this direction. This approach requires thinking about phenotypes in probabilistic terms, a concept that statistical physicists have been applying to particles for a century. Here, I describe PTL and discuss their potential to enlarge our understanding of genotype-phenotype relations. PMID:24315431

Visualization of Arenavirus RNA Species in Individual Cells by Single-Molecule Fluorescence In Situ Hybridization Suggests a Model of Cyclical Infection and Clearance during Persistence.

PubMed

King, Benjamin R; Samacoits, Aubin; Eisenhauer, Philip L; Ziegler, Christopher M; Bruce, Emily A; Zenklusen, Daniel; Zimmer, Christophe; Mueller, Florian; Botten, Jason

2018-06-15

Lymphocytic choriomeningitis mammarenavirus (LCMV) is an enveloped, negative-strand RNA virus that causes serious disease in humans but establishes an asymptomatic, lifelong infection in reservoir rodents. Different models have been proposed to describe how arenaviruses regulate the replication and transcription of their bisegmented, single-stranded RNA genomes, particularly during persistent infection. However, these models were based largely on viral RNA profiling data derived from entire populations of cells. To better understand LCMV replication and transcription at the single-cell level, we established a high-throughput, single-molecule fluorescence in situ hybridization (smFISH) image acquisition and analysis pipeline and examined viral RNA species at discrete time points from virus entry through the late stages of persistent infection in vitro We observed the transcription of viral nucleoprotein and polymerase mRNAs from the incoming S and L segment genomic RNAs, respectively, within 1 h of infection, whereas the transcription of glycoprotein mRNA from the S segment antigenome required ∼4 to 6 h. This confirms the temporal separation of viral gene expression expected due to the ambisense coding strategy of arenaviruses and also suggests that antigenomic RNA contained in virions is not transcriptionally active upon entry. Viral replication and transcription peaked at 36 h postinfection, followed by a progressive loss of viral RNAs over the next several days. During persistence, the majority of cells showed repeating cyclical waves of viral transcription and replication followed by the clearance of viral RNA. Thus, our data support a model of LCMV persistence whereby infected cells can spontaneously clear infection and become reinfected by viral reservoir cells that remain in the population. IMPORTANCE Arenaviruses are human pathogens that can establish asymptomatic, lifelong infections in their rodent reservoirs. Several models have been proposed to explain how arenavirus spread is restricted within host rodents, including the periodic accumulation and loss of replication-competent, but transcriptionally incompetent, viral genomes. A limitation of previous studies was the inability to enumerate viral RNA species at the single-cell level. We developed a high-throughput, smFISH assay and used it to quantitate lymphocytic choriomeningitis mammarenavirus (LCMV) replicative and transcriptional RNA species in individual cells at distinct time points following infection. Our findings support a model whereby productively infected cells can clear infection, including viral RNAs and antigen, and later be reinfected. This information improves our understanding of the timing and possible regulation of LCMV genome replication and transcription during infection. Importantly, the smFISH assay and data analysis pipeline developed here is easily adaptable to other RNA viruses. Copyright © 2018 American Society for Microbiology.

Single crystalline CH 3NH 3PbI 3 self-grown on FTO/TiO 2 substrate for high efficiency perovskite solar cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Jinjin; Kong, Guoli; Chen, Shulin

In this work, we developed an innovative approach to self-grow single crystalline CH 3NH 3PbI 3 directly on polycrystalline FTO/TiO 2 substrate, with which n-i-p type of perovskite solar cells were fabricated. The single crystalline nature of CH 3NH 3PbI 3 has been confirmed by X-ray diffraction and high resolution transmission electron microscopy, and it is observed that they possess smaller optic band gap and longer carrier life time. Highly efficient charge extractions occur at the interface between electron collecting TiO 2 and photo-harvesting CH 3NH 3PbI 3, resulting in a maximum short-circuit current density of 24.40 mA/cm 2. Themore » champion cell possesses a photovoltaic conversion efficiency of 8.78%, and there are still substantial room for further improvement, making it promising for the perovskite solar cell applications.« less

FANTOM5 CAGE profiles of human and mouse samples.

PubMed

Noguchi, Shuhei; Arakawa, Takahiro; Fukuda, Shiro; Furuno, Masaaki; Hasegawa, Akira; Hori, Fumi; Ishikawa-Kato, Sachi; Kaida, Kaoru; Kaiho, Ai; Kanamori-Katayama, Mutsumi; Kawashima, Tsugumi; Kojima, Miki; Kubosaki, Atsutaka; Manabe, Ri-Ichiroh; Murata, Mitsuyoshi; Nagao-Sato, Sayaka; Nakazato, Kenichi; Ninomiya, Noriko; Nishiyori-Sueki, Hiromi; Noma, Shohei; Saijyo, Eri; Saka, Akiko; Sakai, Mizuho; Simon, Christophe; Suzuki, Naoko; Tagami, Michihira; Watanabe, Shoko; Yoshida, Shigehiro; Arner, Peter; Axton, Richard A; Babina, Magda; Baillie, J Kenneth; Barnett, Timothy C; Beckhouse, Anthony G; Blumenthal, Antje; Bodega, Beatrice; Bonetti, Alessandro; Briggs, James; Brombacher, Frank; Carlisle, Ailsa J; Clevers, Hans C; Davis, Carrie A; Detmar, Michael; Dohi, Taeko; Edge, Albert S B; Edinger, Matthias; Ehrlund, Anna; Ekwall, Karl; Endoh, Mitsuhiro; Enomoto, Hideki; Eslami, Afsaneh; Fagiolini, Michela; Fairbairn, Lynsey; Farach-Carson, Mary C; Faulkner, Geoffrey J; Ferrai, Carmelo; Fisher, Malcolm E; Forrester, Lesley M; Fujita, Rie; Furusawa, Jun-Ichi; Geijtenbeek, Teunis B; Gingeras, Thomas; Goldowitz, Daniel; Guhl, Sven; Guler, Reto; Gustincich, Stefano; Ha, Thomas J; Hamaguchi, Masahide; Hara, Mitsuko; Hasegawa, Yuki; Herlyn, Meenhard; Heutink, Peter; Hitchens, Kelly J; Hume, David A; Ikawa, Tomokatsu; Ishizu, Yuri; Kai, Chieko; Kawamoto, Hiroshi; Kawamura, Yuki I; Kempfle, Judith S; Kenna, Tony J; Kere, Juha; Khachigian, Levon M; Kitamura, Toshio; Klein, Sarah; Klinken, S Peter; Knox, Alan J; Kojima, Soichi; Koseki, Haruhiko; Koyasu, Shigeo; Lee, Weonju; Lennartsson, Andreas; Mackay-Sim, Alan; Mejhert, Niklas; Mizuno, Yosuke; Morikawa, Hiromasa; Morimoto, Mitsuru; Moro, Kazuyo; Morris, Kelly J; Motohashi, Hozumi; Mummery, Christine L; Nakachi, Yutaka; Nakahara, Fumio; Nakamura, Toshiyuki; Nakamura, Yukio; Nozaki, Tadasuke; Ogishima, Soichi; Ohkura, Naganari; Ohno, Hiroshi; Ohshima, Mitsuhiro; Okada-Hatakeyama, Mariko; Okazaki, Yasushi; Orlando, Valerio; Ovchinnikov, Dmitry A; Passier, Robert; Patrikakis, Margaret; Pombo, Ana; Pradhan-Bhatt, Swati; Qin, Xian-Yang; Rehli, Michael; Rizzu, Patrizia; Roy, Sugata; Sajantila, Antti; Sakaguchi, Shimon; Sato, Hiroki; Satoh, Hironori; Savvi, Suzana; Saxena, Alka; Schmidl, Christian; Schneider, Claudio; Schulze-Tanzil, Gundula G; Schwegmann, Anita; Sheng, Guojun; Shin, Jay W; Sugiyama, Daisuke; Sugiyama, Takaaki; Summers, Kim M; Takahashi, Naoko; Takai, Jun; Tanaka, Hiroshi; Tatsukawa, Hideki; Tomoiu, Andru; Toyoda, Hiroo; van de Wetering, Marc; van den Berg, Linda M; Verardo, Roberto; Vijayan, Dipti; Wells, Christine A; Winteringham, Louise N; Wolvetang, Ernst; Yamaguchi, Yoko; Yamamoto, Masayuki; Yanagi-Mizuochi, Chiyo; Yoneda, Misako; Yonekura, Yohei; Zhang, Peter G; Zucchelli, Silvia; Abugessaisa, Imad; Arner, Erik; Harshbarger, Jayson; Kondo, Atsushi; Lassmann, Timo; Lizio, Marina; Sahin, Serkan; Sengstag, Thierry; Severin, Jessica; Shimoji, Hisashi; Suzuki, Masanori; Suzuki, Harukazu; Kawai, Jun; Kondo, Naoto; Itoh, Masayoshi; Daub, Carsten O; Kasukawa, Takeya; Kawaji, Hideya; Carninci, Piero; Forrest, Alistair R R; Hayashizaki, Yoshihide

2017-08-29

In the FANTOM5 project, transcription initiation events across the human and mouse genomes were mapped at a single base-pair resolution and their frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled with single-molecule sequencing. Approximately three thousands of samples, consisting of a variety of primary cells, tissues, cell lines, and time series samples during cell activation and development, were subjected to a uniform pipeline of CAGE data production. The analysis pipeline started by measuring RNA extracts to assess their quality, and continued to CAGE library production by using a robotic or a manual workflow, single molecule sequencing, and computational processing to generate frequencies of transcription initiation. Resulting data represents the consequence of transcriptional regulation in each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE profiles, approximately 200,000 and 150,000 peaks for the human and mouse genomes, were identified and annotated to provide precise location of known promoters as well as novel ones, and to quantify their activities.

FANTOM5 CAGE profiles of human and mouse samples

PubMed Central

Noguchi, Shuhei; Arakawa, Takahiro; Fukuda, Shiro; Furuno, Masaaki; Hasegawa, Akira; Hori, Fumi; Ishikawa-Kato, Sachi; Kaida, Kaoru; Kaiho, Ai; Kanamori-Katayama, Mutsumi; Kawashima, Tsugumi; Kojima, Miki; Kubosaki, Atsutaka; Manabe, Ri-ichiroh; Murata, Mitsuyoshi; Nagao-Sato, Sayaka; Nakazato, Kenichi; Ninomiya, Noriko; Nishiyori-Sueki, Hiromi; Noma, Shohei; Saijyo, Eri; Saka, Akiko; Sakai, Mizuho; Simon, Christophe; Suzuki, Naoko; Tagami, Michihira; Watanabe, Shoko; Yoshida, Shigehiro; Arner, Peter; Axton, Richard A.; Babina, Magda; Baillie, J. Kenneth; Barnett, Timothy C.; Beckhouse, Anthony G.; Blumenthal, Antje; Bodega, Beatrice; Bonetti, Alessandro; Briggs, James; Brombacher, Frank; Carlisle, Ailsa J.; Clevers, Hans C.; Davis, Carrie A.; Detmar, Michael; Dohi, Taeko; Edge, Albert S.B.; Edinger, Matthias; Ehrlund, Anna; Ekwall, Karl; Endoh, Mitsuhiro; Enomoto, Hideki; Eslami, Afsaneh; Fagiolini, Michela; Fairbairn, Lynsey; Farach-Carson, Mary C.; Faulkner, Geoffrey J.; Ferrai, Carmelo; Fisher, Malcolm E.; Forrester, Lesley M.; Fujita, Rie; Furusawa, Jun-ichi; Geijtenbeek, Teunis B.; Gingeras, Thomas; Goldowitz, Daniel; Guhl, Sven; Guler, Reto; Gustincich, Stefano; Ha, Thomas J.; Hamaguchi, Masahide; Hara, Mitsuko; Hasegawa, Yuki; Herlyn, Meenhard; Heutink, Peter; Hitchens, Kelly J.; Hume, David A.; Ikawa, Tomokatsu; Ishizu, Yuri; Kai, Chieko; Kawamoto, Hiroshi; Kawamura, Yuki I.; Kempfle, Judith S.; Kenna, Tony J.; Kere, Juha; Khachigian, Levon M.; Kitamura, Toshio; Klein, Sarah; Klinken, S. Peter; Knox, Alan J.; Kojima, Soichi; Koseki, Haruhiko; Koyasu, Shigeo; Lee, Weonju; Lennartsson, Andreas; Mackay-sim, Alan; Mejhert, Niklas; Mizuno, Yosuke; Morikawa, Hiromasa; Morimoto, Mitsuru; Moro, Kazuyo; Morris, Kelly J.; Motohashi, Hozumi; Mummery, Christine L.; Nakachi, Yutaka; Nakahara, Fumio; Nakamura, Toshiyuki; Nakamura, Yukio; Nozaki, Tadasuke; Ogishima, Soichi; Ohkura, Naganari; Ohno, Hiroshi; Ohshima, Mitsuhiro; Okada-Hatakeyama, Mariko; Okazaki, Yasushi; Orlando, Valerio; Ovchinnikov, Dmitry A.; Passier, Robert; Patrikakis, Margaret; Pombo, Ana; Pradhan-Bhatt, Swati; Qin, Xian-Yang; Rehli, Michael; Rizzu, Patrizia; Roy, Sugata; Sajantila, Antti; Sakaguchi, Shimon; Sato, Hiroki; Satoh, Hironori; Savvi, Suzana; Saxena, Alka; Schmidl, Christian; Schneider, Claudio; Schulze-Tanzil, Gundula G.; Schwegmann, Anita; Sheng, Guojun; Shin, Jay W.; Sugiyama, Daisuke; Sugiyama, Takaaki; Summers, Kim M.; Takahashi, Naoko; Takai, Jun; Tanaka, Hiroshi; Tatsukawa, Hideki; Tomoiu, Andru; Toyoda, Hiroo; van de Wetering, Marc; van den Berg, Linda M.; Verardo, Roberto; Vijayan, Dipti; Wells, Christine A.; Winteringham, Louise N.; Wolvetang, Ernst; Yamaguchi, Yoko; Yamamoto, Masayuki; Yanagi-Mizuochi, Chiyo; Yoneda, Misako; Yonekura, Yohei; Zhang, Peter G.; Zucchelli, Silvia; Abugessaisa, Imad; Arner, Erik; Harshbarger, Jayson; Kondo, Atsushi; Lassmann, Timo; Lizio, Marina; Sahin, Serkan; Sengstag, Thierry; Severin, Jessica; Shimoji, Hisashi; Suzuki, Masanori; Suzuki, Harukazu; Kawai, Jun; Kondo, Naoto; Itoh, Masayoshi; Daub, Carsten O.; Kasukawa, Takeya; Kawaji, Hideya; Carninci, Piero; Forrest, Alistair R.R.; Hayashizaki, Yoshihide

2017-01-01

In the FANTOM5 project, transcription initiation events across the human and mouse genomes were mapped at a single base-pair resolution and their frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled with single-molecule sequencing. Approximately three thousands of samples, consisting of a variety of primary cells, tissues, cell lines, and time series samples during cell activation and development, were subjected to a uniform pipeline of CAGE data production. The analysis pipeline started by measuring RNA extracts to assess their quality, and continued to CAGE library production by using a robotic or a manual workflow, single molecule sequencing, and computational processing to generate frequencies of transcription initiation. Resulting data represents the consequence of transcriptional regulation in each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE profiles, approximately 200,000 and 150,000 peaks for the human and mouse genomes, were identified and annotated to provide precise location of known promoters as well as novel ones, and to quantify their activities. PMID:28850106

Single crystalline CH 3NH 3PbI 3 self-grown on FTO/TiO 2 substrate for high efficiency perovskite solar cells

DOE PAGES

Zhao, Jinjin; Kong, Guoli; Chen, Shulin; ...

2017-08-21

In this work, we developed an innovative approach to self-grow single crystalline CH 3NH 3PbI 3 directly on polycrystalline FTO/TiO 2 substrate, with which n-i-p type of perovskite solar cells were fabricated. The single crystalline nature of CH 3NH 3PbI 3 has been confirmed by X-ray diffraction and high resolution transmission electron microscopy, and it is observed that they possess smaller optic band gap and longer carrier life time. Highly efficient charge extractions occur at the interface between electron collecting TiO 2 and photo-harvesting CH 3NH 3PbI 3, resulting in a maximum short-circuit current density of 24.40 mA/cm 2. Themore » champion cell possesses a photovoltaic conversion efficiency of 8.78%, and there are still substantial room for further improvement, making it promising for the perovskite solar cell applications.« less

Study of dynamical process of heat denaturation in optically trapped single microorganisms by near-infrared Raman spectroscopy

NASA Astrophysics Data System (ADS)

Xie, Changan; Li, Yong-qing; Tang, Wei; Newton, Ronald J.

2003-11-01

The development of laser traps has made it possible to investigate single cells and record real-time Raman spectra during a heat-denaturation process when the temperature of the surrounding medium is increased. Large changes in the phenylalanine band (1004 cm-1) of near-infrared spectra between living and heat-treated cells were observed in yeast and Escerichia coli and Enterobacter aerogenes bacteria. This change appears to reflect the change in environment of phenylalanine as proteins within the cells unfold as a result of increasing temperatures. As a comparison, we measured Raman spectra of native and heat-denatured solutions of bovine serum albumin proteins, and a similar change in the phenylalanine band of spectra was observed. In addition, we measured Raman spectra of native and heat-treated solutions of pure phenylalanine molecules; no observable difference in vibrational spectra was observed. These findings may make it possible to study conformational changes in proteins within single cells.

Near-infrared Raman spectroscopy of single optically trapped biological cells

NASA Astrophysics Data System (ADS)

Xie, Changan; Dinno, Mumtaz A.; Li, Yong-Qing

2002-02-01

We report on the development and testing of a compact laser tweezers Raman spectroscopy (LTRS) system. The system combines optical trapping and near-infrared Raman spectroscopy for manipulation and identification of single biological cells in solution. A low-power diode laser at 785 nm was used for both trapping and excitation for Raman spectroscopy of the suspended microscopic particles. The design of the LTRS system provides high sensitivity and permits real-time spectroscopic measurements of the biological sample. The system was calibrated by use of polystyrene microbeads and tested on living blood cells and on both living and dead yeast cells. As expected, different images and Raman spectra were observed for the different cells. The LTRS system may provide a valuable tool for the study of fundamental cellular processes and the diagnosis of cellular disorders.

Pharmacodynamic Modeling of Cell Cycle Effects for Gemcitabine and Trabectedin Combinations in Pancreatic Cancer Cells

PubMed Central

Miao, Xin; Koch, Gilbert; Ait-Oudhia, Sihem; Straubinger, Robert M.; Jusko, William J.

2016-01-01

Combinations of gemcitabine and trabectedin exert modest synergistic cytotoxic effects on two pancreatic cancer cell lines. Here, systems pharmacodynamic (PD) models that integrate cellular response data and extend a prototype model framework were developed to characterize dynamic changes in cell cycle phases of cancer cell subpopulations in response to gemcitabine and trabectedin as single agents and in combination. Extensive experimental data were obtained for two pancreatic cancer cell lines (MiaPaCa-2 and BxPC-3), including cell proliferation rates over 0–120 h of drug exposure, and the fraction of cells in different cell cycle phases or apoptosis. Cell cycle analysis demonstrated that gemcitabine induced cell cycle arrest in S phase, and trabectedin induced transient cell cycle arrest in S phase that progressed to G2/M phase. Over time, cells in the control group accumulated in G0/G1 phase. Systems cell cycle models were developed based on observed mechanisms and were used to characterize both cell proliferation and cell numbers in the sub G1, G0/G1, S, and G2/M phases in the control and drug-treated groups. The proposed mathematical models captured well both single and joint effects of gemcitabine and trabectedin. Interaction parameters were applied to quantify unexplainable drug-drug interaction effects on cell cycle arrest in S phase and in inducing apoptosis. The developed models were able to identify and quantify the different underlying interactions between gemcitabine and trabectedin, and captured well our large datasets in the dimensions of time, drug concentrations, and cellular subpopulations. PMID:27895579

Single-cell isolation by a modular single-cell pipette for RNA-sequencing.

PubMed

Zhang, Kai; Gao, Min; Chong, Zechen; Li, Ying; Han, Xin; Chen, Rui; Qin, Lidong

2016-11-29

Single-cell transcriptome sequencing highly requires a convenient and reliable method to rapidly isolate a live cell into a specific container such as a PCR tube. Here, we report a modular single-cell pipette (mSCP) consisting of three modular components, a SCP-Tip, an air-displacement pipette (ADP), and ADP-Tips, that can be easily assembled, disassembled, and reassembled. By assembling the SCP-Tip containing a hydrodynamic trap, the mSCP can isolate single cells from 5-10 cells per μL of cell suspension. The mSCP is compatible with microscopic identification of captured single cells to finally achieve 100% single-cell isolation efficiency. The isolated live single cells are in submicroliter volumes and well suitable for single-cell PCR analysis and RNA-sequencing. The mSCP possesses merits of convenience, rapidness, and high efficiency, making it a powerful tool to isolate single cells for transcriptome analysis.

Dynamics of different-sized solid-state nanocrystals as tracers for a drug-delivery system in the interstitium of a human tumor xenograft

PubMed Central

Kawai, Masaaki; Higuchi, Hideo; Takeda, Motohiro; Kobayashi, Yoshio; Ohuchi, Noriaki

2009-01-01

Introduction Recent anticancer drugs have been made larger to pass selectively through tumor vessels and stay in the interstitium. Understanding drug movement in association with its size at the single-molecule level and estimating the time needed to reach the targeted organ is indispensable for optimizing drug delivery because single cell-targeted therapy is the ongoing paradigm. This report describes the tracking of single solid nanoparticles in tumor xenografts and the estimation of arrival time. Methods Different-sized nanoparticles measuring 20, 40, and 100 nm were injected into the tail vein of the female Balb/c nu/nu mice bearing human breast cancer on their backs. The movements of the nanoparticles were visualized through the dorsal skin-fold chamber with the high-speed confocal microscopy that we manufactured. Results An analysis of the particle trajectories revealed diffusion to be inversely related to the particle size and position in the tumor, whereas the velocity of the directed movement was related to the position. The difference in the velocity was the greatest for 40-nm particles in the perivascular to the intercellular region: difference = 5.8 nm/s. The arrival time of individual nanoparticles at tumor cells was simulated. The estimated times for the 20-, 40-, and 100-nm particles to reach the tumor cells were 158.0, 218.5, and 389.4 minutes, respectively, after extravasation. Conclusions This result suggests that the particle size can be individually designed for each goal. These data and methods are also important for understanding drug pharmacokinetics. Although this method may be subject to interference by surface molecules attached on the particles, it has the potential to elucidate the pharmacokinetics involved in constructing novel drug-delivery systems involving cell-targeted therapy. PMID:19575785

Listening to the Noise: Random Fluctuations Reveal Gene Network Parameters

NASA Astrophysics Data System (ADS)

Munsky, Brian; Trinh, Brooke; Khammash, Mustafa

2010-03-01

The cellular environment is abuzz with noise originating from the inherent random motion of reacting molecules in the living cell. In this noisy environment, clonal cell populations exhibit cell-to-cell variability that can manifest significant prototypical differences. Noise induced stochastic fluctuations in cellular constituents can be measured and their statistics quantified using flow cytometry, single molecule fluorescence in situ hybridization, time lapse fluorescence microscopy and other single cell and single molecule measurement techniques. We show that these random fluctuations carry within them valuable information about the underlying genetic network. Far from being a nuisance, the ever-present cellular noise acts as a rich source of excitation that, when processed through a gene network, carries its distinctive fingerprint that encodes a wealth of information about that network. We demonstrate that in some cases the analysis of these random fluctuations enables the full identification of network parameters, including those that may otherwise be difficult to measure. We use theoretical investigations to establish experimental guidelines for the identification of gene regulatory networks, and we apply these guideline to experimentally identify predictive models for different regulatory mechanisms in bacteria and yeast.

Ultrasonic Scattering Measurements of a Live Single Cell at 86 MHz

PubMed Central

Lee, Changyang; Jung, Hayong; Lam, Kwok Ho; Yoon, Changhan; Shung, K. Kirk

2016-01-01

Cell separation and sorting techniques have been employed biomedical applications such as cancer diagnosis and cell gene expression analysis. The capability to accurately measure ultrasonic scattering properties from cells is crucial in making an ultrasonic cell sorter a reality if ultrasound scattering is to be used as the sensing mechanism as well. To assess the performance of sensing and identifying live single cells with high-frequency ultrasound, an 86-MHz lithium niobate press-focused single-element acoustic transducer was used in a high-frequency ultrasound scattering measurement system that was custom designed and developed for minimizing noise and allowing better mobility. Peak-to-peak echo amplitude, integrated backscatter (IB) coefficient, spectral parameters including spectral slope and intercept, and midband fit from spectral analysis of the backscattered echoes were measured and calculated from a live single cell of two different types on an agar surface: leukemia cells (K562 cells) and red blood cells (RBCs). The amplitudes of echo signals from K562 cells and RBCs were 48.25 ± 11.98 mVpp and 56.97 ± 7.53 mVpp, respectively. The IB coefficient was −89.39 ± 2.44 dB for K562 cells and −89.00 ± 1.19 dB for RBCs. The spectral slope and intercept were 0.30 ± 0.19 dB/MHz and −56.07 ± 17.17 dB, respectively, for K562 cells and 0.78 ± 0.092 dB/MHz and −98.18 ± 8.80 dB, respectively, for RBCs. Midband fits of K562 cells and RBCs were −31.02 ± 3.04 dB and −33.51 ± 1.55 dB, respectively. Acoustic cellular discrimination via these parameters was tested by Student’s t-test. Their values, except for the IB value, showed statistically significant difference (p < 0.001). This paper reports for the first time that ultrasonic scattering measurements can be made on a live single cell with a highly focused high-frequency ultrasound microbeam at 86 MHz. These results also suggest the feasibility of ultrasonic scattering as a sensing mechanism in the development of ultrasonic cell sorters. PMID:26559626

Dynamic Analysis of Human Natural Killer Cell Response at Single-Cell Resolution in B-Cell Non-Hodgkin Lymphoma.

PubMed

Sarkar, Saheli; Sabhachandani, Pooja; Ravi, Dashnamoorthy; Potdar, Sayalee; Purvey, Sneha; Beheshti, Afshin; Evens, Andrew M; Konry, Tania

2017-01-01

Natural killer (NK) cells are phenotypically and functionally diverse lymphocytes that recognize and kill cancer cells. The susceptibility of target cancer cells to NK cell-mediated cytotoxicity depends on the strength and balance of regulatory (activating/inhibitory) ligands expressed on target cell surface. We performed gene expression arrays to determine patterns of NK cell ligands associated with B-cell non-Hodgkin lymphoma (b-NHL). Microarray analyses revealed significant upregulation of a multitude of NK-activating and costimulatory ligands across varied b-NHL cell lines and primary lymphoma cells, including ULBP1, CD72, CD48, and SLAMF6. To correlate genetic signatures with functional anti-lymphoma activity, we developed a dynamic and quantitative cytotoxicity assay in an integrated microfluidic droplet generation and docking array. Individual NK cells and target lymphoma cells were co-encapsulated in picoliter-volume droplets to facilitate monitoring of transient cellular interactions and NK cell effector outcomes at single-cell level. We identified significant variability in NK-lymphoma cell contact duration, frequency, and subsequent cytolysis. Death of lymphoma cells undergoing single contact with NK cells occurred faster than cells that made multiple short contacts. NK cells also killed target cells in droplets via contact-independent mechanisms that partially relied on calcium-dependent processes and perforin secretion, but not on cytokines (interferon-γ or tumor necrosis factor-α). We extended this technique to characterize functional heterogeneity in cytolysis of primary cells from b-NHL patients. Tumor cells from two diffuse large B-cell lymphoma patients showed similar contact durations with NK cells; primary Burkitt lymphoma cells made longer contacts and were lysed at later times. We also tested the cytotoxic efficacy of NK-92, a continuously growing NK cell line being investigated as an antitumor therapy, using our droplet-based bioassay. NK-92 cells were found to be more efficient in killing b-NHL cells compared with primary NK cells, requiring shorter contacts for faster killing activity. Taken together, our combined genetic and microfluidic analysis demonstrate b-NHL cell sensitivity to NK cell-based cytotoxicity, which was associated with significant heterogeneity in the dynamic interaction at single-cell level.

Tissue aging: the integration of collective and variant responses of cells to entropic forces over time.

PubMed

Todhunter, Michael E; Sayaman, Rosalyn W; Miyano, Masaru; LaBarge, Mark A

2018-06-13

Aging is driven by unavoidable entropic forces, physicochemical in nature, that damage the raw materials that constitute biological systems. Single cells experience and respond to stochastic physicochemical insults that occur either to the cells themselves or to their microenvironment, in a dynamic and reciprocal manner, leading to increased age-related cell-to-cell variation. We will discuss the biological mechanisms that integrate cell-to-cell variation across tissues resulting in stereotypical phenotypes of age. Copyright © 2018 Elsevier Ltd. All rights reserved.

Global Effects of DDX3 Inhibition on Cell Cycle Regulation Identified by a Combined Phosphoproteomics and Single Cell Tracking Approach.

PubMed

Heerma van Voss, Marise R; Kammers, Kai; Vesuna, Farhad; Brilliant, Justin; Bergman, Yehudit; Tantravedi, Saritha; Wu, Xinyan; Cole, Robert N; Holland, Andrew; van Diest, Paul J; Raman, Venu

2018-06-01

DDX3 is an RNA helicase with oncogenic properties. The small molecule inhibitor RK-33 is designed to fit into the ATP binding cleft of DDX3 and hereby block its activity. RK-33 has shown potent activity in preclinical cancer models. However, the mechanism behind the antineoplastic activity of RK-33 remains largely unknown. In this study we used a dual phosphoproteomic and single cell tracking approach to evaluate the effect of RK-33 on cancer cells. MDA-MB-435 cells were treated for 24 hours with RK-33 or vehicle control. Changes in phosphopeptide abundance were analyzed with quantitative mass spectrometry using isobaric mass tags (Tandem Mass Tags). At the proteome level we mainly observed changes in mitochondrial translation, cell division pathways and proteins related to cell cycle progression. Analysis of the phosphoproteome indicated decreased CDK1 activity after RK-33 treatment. To further evaluate the effect of DDX3 inhibition on cell cycle progression over time, we performed timelapse microscopy of Fluorescent Ubiquitin Cell Cycle Indicators labeled cells after RK-33 or siDDX3 exposure. Single cell tracking indicated that DDX3 inhibition resulted in a global delay in cell cycle progression in interphase and mitosis. In addition, we observed an increase in endoreduplication. Overall, we conclude that DDX3 inhibition affects cells in all phases and causes a global cell cycle progression delay. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Rapid high-throughput cloning and stable expression of antibodies in HEK293 cells.

PubMed

Spidel, Jared L; Vaessen, Benjamin; Chan, Yin Yin; Grasso, Luigi; Kline, J Bradford

2016-12-01

Single-cell based amplification of immunoglobulin variable regions is a rapid and powerful technique for cloning antigen-specific monoclonal antibodies (mAbs) for purposes ranging from general laboratory reagents to therapeutic drugs. From the initial screening process involving small quantities of hundreds or thousands of mAbs through in vitro characterization and subsequent in vivo experiments requiring large quantities of only a few, having a robust system for generating mAbs from cloning through stable cell line generation is essential. A protocol was developed to decrease the time, cost, and effort required by traditional cloning and expression methods by eliminating bottlenecks in these processes. Removing the clonal selection steps from the cloning process using a highly efficient ligation-independent protocol and from the stable cell line process by utilizing bicistronic plasmids to generate stable semi-clonal cell pools facilitated an increased throughput of the entire process from plasmid assembly through transient transfections and selection of stable semi-clonal cell pools. Furthermore, the time required by a single individual to clone, express, and select stable cell pools in a high-throughput format was reduced from 4 to 6months to only 4 to 6weeks. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Improved Murine Blastocyst Quality and Development in a Single Culture Medium Compared to Sequential Culture Media

PubMed Central

Hennings, Justin M.; Zimmer, Randall L.; Nabli, Henda; Davis, J. Wade; Sutovsky, Peter; Sutovsky, Miriam; Sharpe-Timms, Kathy L.

2015-01-01

Objective: Validate single versus sequential culture media for murine embryo development. Design: Prospective laboratory experiment. Setting: Assisted Reproduction Laboratory. Animals: Murine embryos. Interventions: Thawed murine zygotes cultured for 3 or 5 days (d3 or d5) in single or sequential embryo culture media developed for human in vitro fertilization. Main Outcome Measures: On d3, zygotes developing to the 8 cell (8C) stage or greater were quantified using 4’,6-diamidino-2-phenylindole (DAPI), and quality was assessed by morphological analysis. On d5, the number of embryos reaching the blastocyst stage was counted. DAPI was used to quantify total nuclei and inner cell mass nuclei. Localization of ubiquitin C-terminal hydrolase L1 (UCHL1) and ubiquitin C-terminal hydrolase L3 (UCHL3) was reference points for evaluating cell quality. Results: Comparing outcomes in single versus to sequential media, the odds of embryos developing to the 8C stage on d3 were 2.34 time greater (P = .06). On d5, more embryos reached the blastocyst stage (P = <.0001), hatched, and had significantly more trophoblast cells (P = .005) contributing to the increased total cell number. Also at d5, localization of distinct cytoplasmic UCHL1 and nuclear UCHL3 was found in high-quality hatching blastocysts. Localization of UCHL1 and UCHL3 was diffuse and inappropriately dispersed throughout the cytoplasm in low-quality nonhatching blastocysts. Conclusions: Single medium yields greater cell numbers, an increased growth rate, and more hatching of murine embryos. Cytoplasmic UCHL1 and nuclear UHCL3 localization patterns were indicative of embryo quality. Our conclusions are limited to murine embryos but one might speculate that single medium may also be more beneficial for human embryo culture. Human embryo studies are needed. PMID:26668049

Improved Murine Blastocyst Quality and Development in a Single Culture Medium Compared to Sequential Culture Media.

PubMed

Hennings, Justin M; Zimmer, Randall L; Nabli, Henda; Davis, J Wade; Sutovsky, Peter; Sutovsky, Miriam; Sharpe-Timms, Kathy L

2016-03-01

Validate single versus sequential culture media for murine embryo development. Prospective laboratory experiment. Assisted Reproduction Laboratory. Murine embryos. Thawed murine zygotes cultured for 3 or 5 days (d3 or d5) in single or sequential embryo culture media developed for human in vitro fertilization. On d3, zygotes developing to the 8 cell (8C) stage or greater were quantified using 4',6-diamidino-2-phenylindole (DAPI), and quality was assessed by morphological analysis. On d5, the number of embryos reaching the blastocyst stage was counted. DAPI was used to quantify total nuclei and inner cell mass nuclei. Localization of ubiquitin C-terminal hydrolase L1 (UCHL1) and ubiquitin C-terminal hydrolase L3 (UCHL3) was reference points for evaluating cell quality. Comparing outcomes in single versus to sequential media, the odds of embryos developing to the 8C stage on d3 were 2.34 time greater (P = .06). On d5, more embryos reached the blastocyst stage (P = <.0001), hatched, and had significantly more trophoblast cells (P = .005) contributing to the increased total cell number. Also at d5, localization of distinct cytoplasmic UCHL1 and nuclear UCHL3 was found in high-quality hatching blastocysts. Localization of UCHL1 and UCHL3 was diffuse and inappropriately dispersed throughout the cytoplasm in low-quality nonhatching blastocysts. Single medium yields greater cell numbers, an increased growth rate, and more hatching of murine embryos. Cytoplasmic UCHL1 and nuclear UHCL3 localization patterns were indicative of embryo quality. Our conclusions are limited to murine embryos but one might speculate that single medium may also be more beneficial for human embryo culture. Human embryo studies are needed. © The Author(s) 2015.

Single-Cell Detection of Secreted Aβ and sAPPα from Human IPSC-Derived Neurons and Astrocytes.

PubMed

Liao, Mei-Chen; Muratore, Christina R; Gierahn, Todd M; Sullivan, Sarah E; Srikanth, Priya; De Jager, Philip L; Love, J Christopher; Young-Pearse, Tracy L

2016-02-03

Secreted factors play a central role in normal and pathological processes in every tissue in the body. The brain is composed of a highly complex milieu of different cell types and few methods exist that can identify which individual cells in a complex mixture are secreting specific analytes. By identifying which cells are responsible, we can better understand neural physiology and pathophysiology, more readily identify the underlying pathways responsible for analyte production, and ultimately use this information to guide the development of novel therapeutic strategies that target the cell types of relevance. We present here a method for detecting analytes secreted from single human induced pluripotent stem cell (iPSC)-derived neural cells and have applied the method to measure amyloid β (Aβ) and soluble amyloid precursor protein-alpha (sAPPα), analytes central to Alzheimer's disease pathogenesis. Through these studies, we have uncovered the dynamic range of secretion profiles of these analytes from single iPSC-derived neuronal and glial cells and have molecularly characterized subpopulations of these cells through immunostaining and gene expression analyses. In examining Aβ and sAPPα secretion from single cells, we were able to identify previously unappreciated complexities in the biology of APP cleavage that could not otherwise have been found by studying averaged responses over pools of cells. This technique can be readily adapted to the detection of other analytes secreted by neural cells, which would have the potential to open new perspectives into human CNS development and dysfunction. We have established a technology that, for the first time, detects secreted analytes from single human neurons and astrocytes. We examine secretion of the Alzheimer's disease-relevant factors amyloid β (Aβ) and soluble amyloid precursor protein-alpha (sAPPα) and present novel findings that could not have been observed without a single-cell analytical platform. First, we identify a previously unappreciated subpopulation that secretes high levels of Aβ in the absence of detectable sAPPα. Further, we show that multiple cell types secrete high levels of Aβ and sAPPα, but cells expressing GABAergic neuronal markers are overrepresented. Finally, we show that astrocytes are competent to secrete high levels of Aβ and therefore may be a significant contributor to Aβ accumulation in the brain. Copyright © 2016 the authors 0270-6474/16/361730-17$15.00/0.

Engineering Yarrowia lipolytica to Simultaneously Produce Lipase and Single Cell Protein from Agro-industrial Wastes for Feed.

PubMed

Yan, Jinyong; Han, Bingnan; Gui, Xiaohua; Wang, Guilong; Xu, Li; Yan, Yunjun; Madzak, Catherine; Pan, Dujie; Wang, Yaofeng; Zha, Genhan; Jiao, Liangcheng

2018-01-15

Lipases are scarcely exploited as feed enzymes in hydrolysis of lipids for increasing energy supply and improving nutrient use efficiency. In this work, we performed homologous overexpression, in vitro characterization and in vivo assessment of a lipase from the yeast Yarrowia lipolytica for feed purpose. Simultaneously, a large amount of yeast cell biomass was produced, for use as single cell protein, a potential protein-rich feed resource. Three kinds of low cost agro-industrial wastes were tested as substrates for simultaneous production of lipase and single cell protein (SCP) as feed additives: sugarcane molasses, waste cooking oil and crude glycerol from biodiesel production. Sugarcane molasses appeared as the most effective cheap medium, allowing production of 16420 U/ml of lipase and 151.2 g/L of single cell protein at 10 liter fermentation scale. In vitro characterization by mimicking a gastro-intestinal environment and determination of essential amino acids of the SCP, and in vivo oral feeding test on fish all revealed that lipase, SCP and their combination were excellent feed additives. Such simultaneous production of this lipase and SCP could address two main concerns of feed industry, poor utilization of lipid and shortage of protein resource at the same time.

Engineering the Intracellular Micro- and Nano-environment via Magnetic Nanoparticles

NASA Astrophysics Data System (ADS)

Tseng, Peter

Single cells, despite being the base unit of living organisms, possess a high degree of hierarchical structure and functional compartmentalization. This complexity exists for good reason: cells must respond efficiently and effectively to its surrounding environment by differentiating, moving, interacting, and more in order to survive or inhabit its role in the larger biological system. At the core of these responses is cellular decision-making. Cells process cues internally and externally from the environment and effect intracellular asymmetry in biochemistry and structure in order to carry out the proper biological responses. Functionalized magnetic particles have shown to be a powerful tool in interacting with biological matter, through either cell or biomolecule sorting, and the activation of biological processes. This dissertation reports on techniques utilizing manipulated magnetic nanoparticles (internalized by cells) to spatially and temporally localize intracellular cues, and examines the resulting asymmetry in biological processes generated by our methods. We first examine patterned micromagnetic elements as a simple strategy of rapidly manipulating magnetic nanoparticles throughout the intracellular space. Silicon or silicon dioxide substrates form the base for electroplated NiFe rods, which are repeated at varying size and pitch. A planarizing resin, initially SU-8, is used as the substrate layer for cellular adhesion. We demonstrate that through the manipulations of a simple external magnet, these micro-fabricated substrates can mediate rapid (under 2 s) and precise (submicron), reversible translation of magnetic nanoparticles through cellular space. Seeding cells on substrates composed of these elements allows simultaneous control of ensembles of nanoparticles over thousands of cells at a time. We believe such substrates could form the basis of magnetically based tools for the activation of biological matter. We further utilize these strategies to generate user-controllable (time-varying and localizable), massively parallel forces on arrays of cells mediated by coalesced ensembles of magnetic nanoparticles. The above process is simplified and adapted for single cell analysis by precisely aligning fibronectin patterned cells to a single flanking micromagnet. The cells are loaded with magnetic-fluorescent nanoparticles, which are then localized to uniform positions at the internal edge of the cell membrane over huge arrays of cells using large external fields, allowing us to conduct composed studies on cellular response to force. By applying forces approaching the yield tension (5 nN / mum) of single cells, we are able to generate highly coordinated responses in cellular behavior. We discover that increasing tension generates highly directed, PAK-dependent leading-edge type filopodia that increase in intensity with rising tension. In addition, we find that our generated forces can simulate cues created during cellular mitosis, as we are consistently able to generate significant (45 to 90 degree) biasing of the metaphase plate during cell division. Large sample size and rapid sample generation also allow us to analyze cells at an unprecedented rate---a single sample can simultaneously stimulate thousands of cells for high statistical accuracy in measurements. We believe these approaches have potential not just as a tool to study single-cell response, but as a means of cell control, potentially through modifying cell movement, division, or differentiation. More generally, once approaches to release nanoparticles from endosomes are implemented, the technique provides a platform to dynamically apply a range of localized stimuli arbitrarily within cells. Through the bioconjugation of proteins, nucleic acids, small molecules, or whole organelles a broad range of questions should be accessible concerning molecular localization and its importance in cell function.

Universal Temporal Profile of Replication Origin Activation in Eukaryotes

NASA Astrophysics Data System (ADS)

Goldar, Arach

2011-03-01

The complete and faithful transmission of eukaryotic genome to daughter cells involves the timely duplication of mother cell's DNA. DNA replication starts at multiple chromosomal positions called replication origin. From each activated replication origin two replication forks progress in opposite direction and duplicate the mother cell's DNA. While it is widely accepted that in eukaryotic organisms replication origins are activated in a stochastic manner, little is known on the sources of the observed stochasticity. It is often associated to the population variability to enter S phase. We extract from a growing Saccharomyces cerevisiae population the average rate of origin activation in a single cell by combining single molecule measurements and a numerical deconvolution technique. We show that the temporal profile of the rate of origin activation in a single cell is similar to the one extracted from a replicating cell population. Taking into account this observation we exclude the population variability as the origin of observed stochasticity in origin activation. We confirm that the rate of origin activation increases in the early stage of S phase and decreases at the latter stage. The population average activation rate extracted from single molecule analysis is in prefect accordance with the activation rate extracted from published micro-array data, confirming therefore the homogeneity and genome scale invariance of dynamic of replication process. All these observations point toward a possible role of replication fork to control the rate of origin activation.

Single Upconversion Nanoparticle-Bacterium Cotrapping for Single-Bacterium Labeling and Analysis.

PubMed

Xin, Hongbao; Li, Yuchao; Xu, Dekang; Zhang, Yueli; Chen, Chia-Hung; Li, Baojun

2017-04-01

Detecting and analyzing pathogenic bacteria in an effective and reliable manner is crucial for the diagnosis of acute bacterial infection and initial antibiotic therapy. However, the precise labeling and analysis of bacteria at the single-bacterium level are a technical challenge but very important to reveal important details about the heterogeneity of cells and responds to environment. This study demonstrates an optical strategy for single-bacterium labeling and analysis by the cotrapping of single upconversion nanoparticles (UCNPs) and bacteria together. A single UCNP with an average size of ≈120 nm is first optically trapped. Both ends of a single bacterium are then trapped and labeled with single UCNPs emitting green light. The labeled bacterium can be flexibly moved to designated locations for further analysis. Signals from bacteria of different sizes are detected in real time for single-bacterium analysis. This cotrapping method provides a new approach for single-pathogenic-bacterium labeling, detection, and real-time analysis at the single-particle and single-bacterium level. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fluorescence lifetime imaging of induced pluripotent stem cells

NASA Astrophysics Data System (ADS)

Uchugonova, Aisada; Batista, Ana; König, Karsten

2014-02-01

The multiphoton FLIM tomograph MPTflex with its flexible scan head, articulated arm, and the tunable femtosecond laser source was employed to study cell monolayers and 3D cell clusters. FLIM was performed with 250 ps temporal resolution and submicron special resolution using time-correlated single photon counting. The autofluorescence based on NAD(P)H and flavins/flavoproteins has been measured in mouse embryonic fibroblasts, induced pluripotent stem cells (iPS cells) originated from mouse embryonic fibroblasts and non-proliferative mouse embryonic fibroblasts.

Evaluation of Biofilms and the Effects of Biocides Thereon

NASA Technical Reports Server (NTRS)

Pierson, Duane L. (Inventor); Koenig, David W. (Inventor); Mishra, Saroj K. (Inventor)

2002-01-01

Biofilm formation is monitored by real-time continuous measurement. Images are formed of sessile cells on a surface and planktonic cells adjacent the surface. The attachment of cells to the surface is measured and quantitated, and sessile and planktonic cells are distinguished using image processing techniques. Single cells as well as colonies are monitored on or adjacent a variety of substrates. Flowing streams may be monitored. The effects of biocides on biofilms commonly isolated from recyclable water systems are measured.

A new effective assay to detect antimicrobial activity of filamentous fungi.

PubMed

Pereira, Eric; Santos, Ana; Reis, Francisca; Tavares, Rui M; Baptista, Paula; Lino-Neto, Teresa; Almeida-Aguiar, Cristina

2013-01-15

The search for new antimicrobial compounds and the optimization of production methods turn the use of antimicrobial susceptibility tests a routine. The most frequently used methods are based on agar diffusion assays or on dilution in agar or broth. For filamentous fungi, the most common antimicrobial activity detection methods comprise the co-culture of two filamentous fungal strains or the use of fungal extracts to test against single-cell microorganisms. Here we report a rapid, effective and reproducible assay to detect fungal antimicrobial activity against single-cell microorganisms. This method allows an easy way of performing a fast antimicrobial screening of actively growing fungi directly against yeast. Because it makes use of an actively growing mycelium, this bioassay also provides a way for studying the production dynamics of antimicrobial compounds by filamentous fungi. The proposed assay is less time consuming and introduces the innovation of allowing the direct detection of fungal antimicrobial properties against single cell microorganisms without the prior isolation of the active substance(s). This is particularly useful when performing large screenings for fungal antimicrobial activity. With this bioassay, antimicrobial activity of Hypholoma fasciculare against yeast species was observed for the first time. Copyright © 2012 Elsevier GmbH. All rights reserved.

Induction of Thioguanine- and Ouabain-Resistant Mutants and Single-Strand Breaks in the DNA of Chinese Hamster Ovary Cells by 3H-Thymidine

PubMed Central

Cleaver, James E.

1977-01-01

Cultured Chinese hamster cells were labeled with 6-3H-thymidine or 5-methyl-3H-thymidine and allowed to accumulate damage from 3H decays for various periods of time while frozen. The frequencies of cells resistant to 6-thioguanine or ouabain and the amount of DNA damage (i.e., number of single-strand breaks) were determined and compared with the mutation frequencies resulting from X and ultraviolet light irradiation. Whereas 3H decays and X rays made only 6-thioguanine-resistant mutants, ultraviolet light made both 6-thioguanine- and ouabain-resistant mutants. 3H decays originating at the 6 position were two to three times as effective as decays at the 5-methyl position in making drug-resistant mutants, but decays at both sites were equally effective in making single-strand breaks. Mutants and strand breaks produced by beta irradiation of the nucleus probably are the same irrespective of the site of the decay in thymine; these results indicate that the local transmutation effects of 3H decay produce more mutations when they occur at the 6 position than at the 5-methyl position. PMID:914028

Intracellular pH Response to Weak Acid Stress in Individual Vegetative Bacillus subtilis Cells

PubMed Central

Pandey, Rachna; Vischer, Norbert O. E.; Smelt, Jan P. P. M.; van Beilen, Johan W. A.; Ter Beek, Alexander; De Vos, Winnok H.; Manders, Erik M. M.

2016-01-01

ABSTRACT Intracellular pH (pHi) critically affects bacterial cell physiology. Hence, a variety of food preservation strategies are aimed at perturbing pHi homeostasis. Unfortunately, accurate pHi quantification with existing methods is suboptimal, since measurements are averages across populations of cells, not taking into account interindividual heterogeneity. Yet, physiological heterogeneity in isogenic populations is well known to be responsible for differences in growth and division kinetics of cells in response to external stressors. To assess in this context the behavior of intracellular acidity, we have developed a robust method to quantify pHi at single-cell levels in Bacillus subtilis. Bacilli spoil food, cause disease, and are well known for their ability to form highly stress-resistant spores. Using an improved version of the genetically encoded ratiometric pHluorin (IpHluorin), we have quantified pHi in individual B. subtilis cells, cultured at an external pH of 6.4, in the absence or presence of weak acid stresses. In the presence of 3 mM potassium sorbate, a decrease in pHi and an increase in the generation time of growing cells were observed. Similar effects were observed when cells were stressed with 25 mM potassium acetate. Time-resolved analysis of individual bacteria in growing colonies shows that after a transient pH decrease, long-term pH evolution is highly cell dependent. The heterogeneity at the single-cell level shows the existence of subpopulations that might be more resistant and contribute to population survival. Our approach contributes to an understanding of pHi regulation in individual bacteria and may help scrutinizing effects of existing and novel food preservation strategies. IMPORTANCE This study shows how the physiological response to commonly used weak organic acid food preservatives, such as sorbic and acetic acids, can be measured at the single-cell level. These data are key to coupling often-observed single-cell heterogeneous growth behavior upon the addition of weak organic acid food preservatives. Generally, these data are gathered in the form of plate counting of samples incubated with the acids. Here, we visualize the underlying heterogeneity in cellular pH homeostasis, opening up avenues for mechanistic analyses of the heterogeneity in the weak acid stress response. Thus, microbial risk assessment can become more robust, widening the scope of use of these well-known weak organic acid food preservatives. PMID:27565617

Visualization of metallodrugs in single cells by secondary ion mass spectrometry imaging.

PubMed

Wu, Kui; Jia, Feifei; Zheng, Wei; Luo, Qun; Zhao, Yao; Wang, Fuyi

2017-07-01

Secondary ion mass spectrometry, including nanoscale secondary ion mass spectrometry (NanoSIMS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS), has emerged as a powerful tool for biological imaging, especially for single cell imaging. SIMS imaging can provide information on subcellular distribution of endogenous and exogenous chemicals, including metallodrugs, from membrane through to cytoplasm and nucleus without labeling, and with high spatial resolution and chemical specificity. In this mini-review, we summarize recent progress in the field of SIMS imaging, particularly in the characterization of the subcellular distribution of metallodrugs. We anticipate that the SIMS imaging method will be widely applied to visualize subcellular distributions of drugs and drug candidates in single cells, exerting significant influence on early drug evaluation and metabolism in medicinal and pharmaceutical chemistry. Recent progress of SIMS applications in characterizing the subcellular distributions of metallodrugs was summarized.

Imaging the beating heart in the mouse using intravital microscopy techniques

PubMed Central

Vinegoni, Claudio; Aguirre, Aaron D; Lee, Sungon; Weissleder, Ralph

2017-01-01

Real-time microscopic imaging of moving organs at single-cell resolution represents a major challenge in studying complex biology in living systems. Motion of the tissue from the cardiac and respiratory cycles severely limits intravital microscopy by compromising ultimate spatial and temporal imaging resolution. However, significant recent advances have enabled single-cell resolution imaging to be achieved in vivo. In this protocol, we describe experimental procedures for intravital microscopy based on a combination of thoracic surgery, tissue stabilizers and acquisition gating methods, which enable imaging at the single-cell level in the beating heart in the mouse. Setup of the model is typically completed in 1 h, which allows 2 h or more of continuous cardiac imaging. This protocol can be readily adapted for the imaging of other moving organs, and it will therefore broadly facilitate in vivo high-resolution microscopy studies. PMID:26492138

Rapid determination of cell mass and density using digitally controlled electric field in a microfluidic chip.

PubMed

Zhao, Yuliang; Lai, Hok Sum Sam; Zhang, Guanglie; Lee, Gwo-Bin; Li, Wen Jung

2014-11-21

The density of a single cell is a fundamental property of cells. Cells in the same cycle phase have similar volume, but the differences in their mass and density could elucidate each cell's physiological state. Here we report a novel technique to rapidly measure the density and mass of a single cell using an optically induced electrokinetics (OEK) microfluidic platform. Presently, single cellular mass and density measurement devices require a complicated fabrication process and their output is not scalable, i.e., it is extremely difficult to measure the mass and density of a large quantity of cells rapidly. The technique reported here operates on a principle combining sedimentation theory, computer vision, and microparticle manipulation techniques in an OEK microfluidic platform. We will show in this paper that this technique enables the measurement of single-cell volume, density, and mass rapidly and accurately in a repeatable manner. The technique is also scalable - it allows simultaneous measurement of volume, density, and mass of multiple cells. Essentially, a simple time-controlled projected light pattern is used to illuminate the selected area on the OEK microfluidic chip that contains cells to lift the cells to a particular height above the chip's surface. Then, the cells are allowed to "free fall" to the chip's surface, with competing buoyancy, gravitational, and fluidic drag forces acting on the cells. By using a computer vision algorithm to accurately track the motion of the cells and then relate the cells' motion trajectory to sedimentation theory, the volume, mass, and density of each cell can be rapidly determined. A theoretical model of micro-sized spheres settling towards an infinite plane in a microfluidic environment is first derived and validated experimentally using standard micropolystyrene beads to demonstrate the viability and accuracy of this new technique. Next, we show that the yeast cell volume, mass, and density could be rapidly determined using this technology, with results comparable to those using the existing method suspended microchannel resonator.

Validation of a device for the active manipulation of the tumor microenvironment during intravital imaging

PubMed Central

Williams, James K.; Entenberg, David; Wang, Yarong; Avivar-Valderas, Alvaro; Padgen, Michael; Clark, Ashley; Aguirre-Ghiso, Julio A.; Castracane, James; Condeelis, John S.

2016-01-01

ABSTRACT The tumor microenvironment is recognized as playing a significant role in the behavior of tumor cells and their progression to metastasis. However, tools to manipulate the tumor microenvironment directly, and image the consequences of this manipulation with single cell resolution in real time in vivo, are lacking. We describe here a method for the direct, local manipulation of microenvironmental parameters through the use of an implantable Induction Nano Intravital Device (iNANIVID) and simultaneous in vivo visualization of the results at single-cell resolution. As a proof of concept, we deliver both a sustained dose of EGF to tumor cells while intravital imaging their chemotactic response as well as locally induce hypoxia in defined microenvironments in solid tumors. PMID:27790386

Measurement of In Vitro Single Cell Temperature by Novel Thermocouple Nanoprobe in Acute Lung Injury Models.

PubMed

Wang, Xing; Chen, Qiuhua; Tian, Wenjuan; Wang, Jianqing; Cheng, Lu; Lu, Jun; Chen, Mingqi; Pei, Yinhao; Li, Can; Chen, Gong; Gu, Ning

2017-01-01

Energy metabolism may alter pattern differences in acute lung injury (ALI) as one of the causes but the detailed features at single-cellular level remain unclear. Changes in intercellular temperature and adenosine triphosphate (ATP) concentration within the single cell may help to understand the role of energy metabolism in causing ALI. ALI in vitro models were established by treating mice lung epithelial (MLE-12) cells with lipopolysaccharide (LPS), hydrogen peroxide (H2O2), hydrochloric acid (HCl) and cobalt chloride (CoCl2, respectively. 100 nm micro thermocouple probe (TMP) was inserted into the cytosol by micromanipulation system and thermoelectric readings were recorded to calculate the intracellular temperature based on standard curve. The total ATP contents for the MLE-12 cells were evaluated at different time intervals after treatments. A significant increase of intracellular temperature was observed after 10 or 20 μg/L LPS and HCl treatments. The HCl increased the temperature in a dose-dependent manner. On the contrary, H2O2 induced a significant decline of intracellular temperature after treatment. No significant difference in intracellular temperature was observed after CoCl2 exposure. The intracellular ATP levels decreased in a time-dependent manner after treatment with H2O2 and HCl, while the LPS and CoCl2 had no significant effect on ATP levels. The intracellular temperature responses varied in different ALI models. The concentration of ATP in the MLE-12 cells played part in the intracellular temperature changes. No direct correlation was observed between the intracellular temperature and concentration of ATP in the MLE-12 cells.

Differentiation of K562 cells under ELF-EMF applied at different time courses.

PubMed

Ayşe, Inhan-Garip; Zafer, Akan; Sule, Oncul; Işil, Işal-Turgut; Kalkan, Tunaya

2010-08-01

The time-course of ELF-EMF application to biological systems is thought to be an important parameter determining the physiological outcome. This study investigated the effect of ELF-EMF on the differentiation of K562 cells at different time courses. ELF-EMF (50 Hz, 5 mT, 1 h) was applied at two different time-courses; first at the onset of hemin induction for 1 h, and second, daily 1 h for four days. While single exposure to ELF-EMF resulted in a decrease in differentiation, ELF-EMF applied everyday for 1 h caused an increase in differentiation. The effect of co-stressors, magnesium, and heat-shock was also determined and similar results were obtained. ELF-EMF increased ROS levels in K562 cells not treated with hemin, however did not change ROS levels of hemin treated cells indicating that ROS was not the cause. Overall, these results imply that the time-course of application is an important parameter determining the physiological response of cells to ELF-EMF.

Estimation of life times and diffusion distances of radicals involved in x- ray-induced DNA strand breaks of killing of mammalian cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roots, R; Okada, S

1975-11-01

We have used a mammalian tissue culture system to calculate the life times and diffusion distances in DNA scissions as well as cell killing for the three main products of water radiolysis: OH, H, and e$sup -$/sub aq/. Using various alcohols as radical scavengers, the average life time for OH in DNA single-strand breaks was calculated to be about 4 x 10$sup -9$ sec. Using the same data and published rate constants, the apparent life time of H atoms was calculated to vary from about 2 x 10$sup -7$ to 4 x 10$sup -6$ sec and, similarly, the calculated lifemore » time of the hydrated electron was found to vary more than was the case for OH. From these life times, the radical diffusion distances were estimated to be approximately 60 A for OH, which is reasonable, but the values for both H and e$sup -$/sub aq/ were unrealistically large, i.e., 880 to 4040 A for H and 9590 to 19,810 A for e$sup -$/sub aq/. In cell killing, the OH radical life time was estimated to be about 8.7 x 10$sup -9$ sec which gives an average diffusion distance for this radical of about 93 A. Our data support the idea that OH is the radical species primarily responsible for the indirect effect in radiation injury measured as DNA single-strand breaks or cell killing, and that H and e$sup -$/sub aq/ are not significantly involved. (auth)« less

Finite Element Analysis of Single Cell Stiffness Measurements Using PZT-Integrated Buckling Nanoneedles

PubMed Central

Rad, Maryam Alsadat; Tijjani, Auwal Shehu; Ahmad, Mohd Ridzuan; Auwal, Shehu Muhammad

2016-01-01

This paper proposes a new technique for real-time single cell stiffness measurement using lead zirconate titanate (PZT)-integrated buckling nanoneedles. The PZT and the buckling part of the nanoneedle have been modelled and validated using the ABAQUS software. The two parts are integrated together to function as a single unit. After calibration, the stiffness, Young’s modulus, Poisson’s ratio and sensitivity of the PZT-integrated buckling nanoneedle have been determined to be 0.7100 N·m−1, 123.4700 GPa, 0.3000 and 0.0693 V·m·N−1, respectively. Three Saccharomyces cerevisiae cells have been modelled and validated based on compression tests. The average global stiffness and Young’s modulus of the cells are determined to be 10.8867 ± 0.0094 N·m−1 and 110.7033 ± 0.0081 MPa, respectively. The nanoneedle and the cell have been assembled to measure the local stiffness of the single Saccharomyces cerevisiae cells The local stiffness, Young’s modulus and PZT output voltage of the three different size Saccharomyces cerevisiae have been determined at different environmental conditions. We investigated that, at low temperature the stiffness value is low to adapt to the change in the environmental condition. As a result, Saccharomyces cerevisiae becomes vulnerable to viral and bacterial attacks. Therefore, the proposed technique will serve as a quick and accurate process to diagnose diseases at early stage in a cell for effective treatment. PMID:28025571

Confocal Raman imaging for cancer cell classification

NASA Astrophysics Data System (ADS)

Mathieu, Evelien; Van Dorpe, Pol; Stakenborg, Tim; Liu, Chengxun; Lagae, Liesbet

2014-05-01

We propose confocal Raman imaging as a label-free single cell characterization method that can be used as an alternative for conventional cell identification techniques that typically require labels, long incubation times and complex sample preparation. In this study it is investigated whether cancer and blood cells can be distinguished based on their Raman spectra. 2D Raman scans are recorded of 114 single cells, i.e. 60 breast (MCF-7), 5 cervix (HeLa) and 39 prostate (LNCaP) cancer cells and 10 monocytes (from healthy donors). For each cell an average spectrum is calculated and principal component analysis is performed on all average cell spectra. The main features of these principal components indicate that the information for cell identification based on Raman spectra mainly comes from the fatty acid composition in the cell. Based on the second and third principal component, blood cells could be distinguished from cancer cells; and prostate cancer cells could be distinguished from breast and cervix cancer cells. However, it was not possible to distinguish breast and cervix cancer cells. The results obtained in this study, demonstrate the potential of confocal Raman imaging for cell type classification and identification purposes.

Inertial picobalance reveals fast mass fluctuations in mammalian cells

NASA Astrophysics Data System (ADS)

Martínez-Martín, David; Fläschner, Gotthold; Gaub, Benjamin; Martin, Sascha; Newton, Richard; Beerli, Corina; Mercer, Jason; Gerber, Christoph; Müller, Daniel J.

2017-10-01

The regulation of size, volume and mass in living cells is physiologically important, and dysregulation of these parameters gives rise to many diseases. Cell mass is largely determined by the amount of water, proteins, lipids, carbohydrates and nucleic acids present in a cell, and is tightly linked to metabolism, proliferation and gene expression. Technologies have emerged in recent years that make it possible to track the masses of single suspended cells and adherent cells. However, it has not been possible to track individual adherent cells in physiological conditions at the mass and time resolutions required to observe fast cellular dynamics. Here we introduce a cell balance (a ‘picobalance’), based on an optically excited microresonator, that measures the total mass of single or multiple adherent cells in culture conditions over days with millisecond time resolution and picogram mass sensitivity. Using our technique, we observe that the mass of living mammalian cells fluctuates intrinsically by around one to four per cent over timescales of seconds throughout the cell cycle. Perturbation experiments link these mass fluctuations to the basic cellular processes of ATP synthesis and water transport. Furthermore, we show that growth and cell cycle progression are arrested in cells infected with vaccinia virus, but mass fluctuations continue until cell death. Our measurements suggest that all living cells show fast and subtle mass fluctuations throughout the cell cycle. As our cell balance is easy to handle and compatible with fluorescence microscopy, we anticipate that our approach will contribute to the understanding of cell mass regulation in various cell states and across timescales, which is important in areas including physiology, cancer research, stem-cell differentiation and drug discovery.

Detecting bacteria and Determining Their Susceptibility to Antibiotics by Stochastic Confinement in Nanoliter Droplets using Plug-Based Microfluidics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boedicker, J.; Li, L; Kline, T

2008-01-01

This article describes plug-based microfluidic technology that enables rapid detection and drug susceptibility screening of bacteria in samples, including complex biological matrices, without pre-incubation. Unlike conventional bacterial culture and detection methods, which rely on incubation of a sample to increase the concentration of bacteria to detectable levels, this method confines individual bacteria into droplets nanoliters in volume. When single cells are confined into plugs of small volume such that the loading is less than one bacterium per plug, the detection time is proportional to plug volume. Confinement increases cell density and allows released molecules to accumulate around the cell, eliminatingmore » the pre-incubation step and reducing the time required to detect the bacteria. We refer to this approach as stochastic confinement. Using the microfluidic hybrid method, this technology was used to determine the antibiogram - or chart of antibiotic sensitivity - of methicillin-resistant Staphylococcus aureus (MRSA) to many antibiotics in a single experiment and to measure the minimal inhibitory concentration (MIC) of the drug cefoxitin (CFX) against this strain. In addition, this technology was used to distinguish between sensitive and resistant strains of S. aureus in samples of human blood plasma. High-throughput microfluidic techniques combined with single-cell measurements also enable multiple tests to be performed simultaneously on a single sample containing bacteria. This technology may provide a method of rapid and effective patient-specific treatment of bacterial infections and could be extended to a variety of applications that require multiple functional tests of bacterial samples on reduced timescales.« less

Collective Cell Behavior in Mechanosensing of Substrate Thickness.

PubMed

Tusan, Camelia G; Man, Yu-Hin; Zarkoob, Hoda; Johnston, David A; Andriotis, Orestis G; Thurner, Philipp J; Yang, Shoufeng; Sander, Edward A; Gentleman, Eileen; Sengers, Bram G; Evans, Nicholas D

2018-06-05

Extracellular matrix stiffness has a profound effect on the behavior of many cell types. Adherent cells apply contractile forces to the material on which they adhere and sense the resistance of the material to deformation-its stiffness. This is dependent on both the elastic modulus and the thickness of the material, with the corollary that single cells are able to sense underlying stiff materials through soft hydrogel materials at low (<10 μm) thicknesses. Here, we hypothesized that cohesive colonies of cells exert more force and create more hydrogel deformation than single cells, therefore enabling them to mechanosense more deeply into underlying materials than single cells. To test this, we modulated the thickness of soft (1 kPa) elastic extracellular-matrix-functionalized polyacrylamide hydrogels adhered to glass substrates and allowed colonies of MG63 cells to form on their surfaces. Cell morphology and deformations of fluorescent fiducial-marker-labeled hydrogels were quantified by time-lapse fluorescence microscopy imaging. Single-cell spreading increased with respect to decreasing hydrogel thickness, with data fitting to an exponential model with half-maximal response at a thickness of 3.2 μm. By quantifying cell area within colonies of defined area, we similarly found that colony-cell spreading increased with decreasing hydrogel thickness but with a greater half-maximal response at 54 μm. Depth-sensing was dependent on Rho-associated protein kinase-mediated cellular contractility. Surface hydrogel deformations were significantly greater on thick hydrogels compared to thin hydrogels. In addition, deformations extended greater distances from the periphery of colonies on thick hydrogels compared to thin hydrogels. Our data suggest that by acting collectively, cells mechanosense rigid materials beneath elastic hydrogels at greater depths than individual cells. This raises the possibility that the collective action of cells in colonies or sheets may allow cells to sense structures of differing material properties at comparatively large distances. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Single cell adhesion assay using computer controlled micropipette.

PubMed

Salánki, Rita; Hős, Csaba; Orgovan, Norbert; Péter, Beatrix; Sándor, Noémi; Bajtay, Zsuzsa; Erdei, Anna; Horvath, Robert; Szabó, Bálint

2014-01-01

Cell adhesion is a fundamental phenomenon vital for all multicellular organisms. Recognition of and adhesion to specific macromolecules is a crucial task of leukocytes to initiate the immune response. To gain statistically reliable information of cell adhesion, large numbers of cells should be measured. However, direct measurement of the adhesion force of single cells is still challenging and today's techniques typically have an extremely low throughput (5-10 cells per day). Here, we introduce a computer controlled micropipette mounted onto a normal inverted microscope for probing single cell interactions with specific macromolecules. We calculated the estimated hydrodynamic lifting force acting on target cells by the numerical simulation of the flow at the micropipette tip. The adhesion force of surface attached cells could be accurately probed by repeating the pick-up process with increasing vacuum applied in the pipette positioned above the cell under investigation. Using the introduced methodology hundreds of cells adhered to specific macromolecules were measured one by one in a relatively short period of time (∼30 min). We blocked nonspecific cell adhesion by the protein non-adhesive PLL-g-PEG polymer. We found that human primary monocytes are less adherent to fibrinogen than their in vitro differentiated descendants: macrophages and dendritic cells, the latter producing the highest average adhesion force. Validation of the here introduced method was achieved by the hydrostatic step-pressure micropipette manipulation technique. Additionally the result was reinforced in standard microfluidic shear stress channels. Nevertheless, automated micropipette gave higher sensitivity and less side-effect than the shear stress channel. Using our technique, the probed single cells can be easily picked up and further investigated by other techniques; a definite advantage of the computer controlled micropipette. Our experiments revealed the existence of a sub-population of strongly fibrinogen adherent cells appearing in macrophages and highly represented in dendritic cells, but not observed in monocytes.

Single Cell Adhesion Assay Using Computer Controlled Micropipette

PubMed Central

Salánki, Rita; Hős, Csaba; Orgovan, Norbert; Péter, Beatrix; Sándor, Noémi; Bajtay, Zsuzsa; Erdei, Anna; Horvath, Robert; Szabó, Bálint

2014-01-01

Cell adhesion is a fundamental phenomenon vital for all multicellular organisms. Recognition of and adhesion to specific macromolecules is a crucial task of leukocytes to initiate the immune response. To gain statistically reliable information of cell adhesion, large numbers of cells should be measured. However, direct measurement of the adhesion force of single cells is still challenging and today’s techniques typically have an extremely low throughput (5–10 cells per day). Here, we introduce a computer controlled micropipette mounted onto a normal inverted microscope for probing single cell interactions with specific macromolecules. We calculated the estimated hydrodynamic lifting force acting on target cells by the numerical simulation of the flow at the micropipette tip. The adhesion force of surface attached cells could be accurately probed by repeating the pick-up process with increasing vacuum applied in the pipette positioned above the cell under investigation. Using the introduced methodology hundreds of cells adhered to specific macromolecules were measured one by one in a relatively short period of time (∼30 min). We blocked nonspecific cell adhesion by the protein non-adhesive PLL-g-PEG polymer. We found that human primary monocytes are less adherent to fibrinogen than their in vitro differentiated descendants: macrophages and dendritic cells, the latter producing the highest average adhesion force. Validation of the here introduced method was achieved by the hydrostatic step-pressure micropipette manipulation technique. Additionally the result was reinforced in standard microfluidic shear stress channels. Nevertheless, automated micropipette gave higher sensitivity and less side-effect than the shear stress channel. Using our technique, the probed single cells can be easily picked up and further investigated by other techniques; a definite advantage of the computer controlled micropipette. Our experiments revealed the existence of a sub-population of strongly fibrinogen adherent cells appearing in macrophages and highly represented in dendritic cells, but not observed in monocytes. PMID:25343359

In Vivo Single-Cell Fluorescence and Size Scaling of Phytoplankton Chlorophyll Content.

PubMed

Álvarez, Eva; Nogueira, Enrique; López-Urrutia, Ángel

2017-04-01

In unicellular phytoplankton, the size scaling exponent of chlorophyll content per cell decreases with increasing light limitation. Empirical studies have explored this allometry by combining data from several species, using average values of pigment content and cell size for each species. The resulting allometry thus includes phylogenetic and size scaling effects. The possibility of measuring single-cell fluorescence with imaging-in-flow cytometry devices allows the study of the size scaling of chlorophyll content at both the inter- and intraspecific levels. In this work, the changing allometry of chlorophyll content was estimated for the first time for single phytoplankton populations by using data from a series of incubations with monocultures exposed to different light levels. Interspecifically, our experiments confirm previous modeling and experimental results of increasing size scaling exponents with increasing irradiance. A similar pattern was observed intraspecifically but with a larger variability in size scaling exponents. Our results show that size-based processes and geometrical approaches explain variations in chlorophyll content. We also show that the single-cell fluorescence measurements provided by imaging-in-flow devices can be applied to field samples to understand the changes in the size dependence of chlorophyll content in response to environmental variables affecting primary production. IMPORTANCE The chlorophyll concentrations in phytoplankton register physiological adjustments in cellular pigmentation arising mainly from changes in light conditions. The extent of these adjustments is constrained by the size of the phytoplankton cells, even within single populations. Hence, variations in community chlorophyll derived from photoacclimation are also dependent on the phytoplankton size distribution. Copyright © 2017 American Society for Microbiology.

In Vivo Single-Cell Fluorescence and Size Scaling of Phytoplankton Chlorophyll Content

PubMed Central

Nogueira, Enrique; López-Urrutia, Ángel

2017-01-01

ABSTRACT In unicellular phytoplankton, the size scaling exponent of chlorophyll content per cell decreases with increasing light limitation. Empirical studies have explored this allometry by combining data from several species, using average values of pigment content and cell size for each species. The resulting allometry thus includes phylogenetic and size scaling effects. The possibility of measuring single-cell fluorescence with imaging-in-flow cytometry devices allows the study of the size scaling of chlorophyll content at both the inter- and intraspecific levels. In this work, the changing allometry of chlorophyll content was estimated for the first time for single phytoplankton populations by using data from a series of incubations with monocultures exposed to different light levels. Interspecifically, our experiments confirm previous modeling and experimental results of increasing size scaling exponents with increasing irradiance. A similar pattern was observed intraspecifically but with a larger variability in size scaling exponents. Our results show that size-based processes and geometrical approaches explain variations in chlorophyll content. We also show that the single-cell fluorescence measurements provided by imaging-in-flow devices can be applied to field samples to understand the changes in the size dependence of chlorophyll content in response to environmental variables affecting primary production. IMPORTANCE The chlorophyll concentrations in phytoplankton register physiological adjustments in cellular pigmentation arising mainly from changes in light conditions. The extent of these adjustments is constrained by the size of the phytoplankton cells, even within single populations. Hence, variations in community chlorophyll derived from photoacclimation are also dependent on the phytoplankton size distribution. PMID:28115378

Impact of long term cryopreservation on single umbilical cord blood transplant outcomes

PubMed Central

Mitchell, R.; Wagner, J.E.; Brunstein, C.G.; Cao, Q.; McKenna, D.H.; Lund, T.C.; Verneris, M.R.

2015-01-01

Umbilical cord blood (UCB) has the advantage of being collected and cryopreserved for years prior to use. In vitro or in murine models suggest that the duration of storage does not affect UCB progenitor cell performance, however the impact of UCB age on clinical outcomes has not been definitely defined. This study sought to determine the effect of UCB unit cryopreservation time on hematopoietic potency. We analyzed 288 single UCB units used for transplantation from 1992–2013, with unit cryopreservation time ranging from 0.08 to 11.07 years. UCB unit post thaw characteristics were examined, including percent recovery of total nucleated cells (TNC). The number of years the UCB unit spent in cryopreservation had no impact on TNC recovery nor UCB unit post-thaw viability. Duration of cryopreservation also had no impact on neutrophil or platelet engraftment in single UCB transplants. These results show that UCB units can undergo cryopreservation for at least 10 years with no impact on clinical outcomes. PMID:25262882

Viscoelastic Properties Measurement of Human Lymphocytes by Atomic Force Microscopy Based on Magnetic Beads Cell Isolation.

PubMed

Mi Li; Lianqing Liu; Xiubin Xiao; Ning Xi; Yuechao Wang

2016-07-01

Cell mechanics has been proved to be an effective biomarker for indicating cellular states. The advent of atomic force microscopy (AFM) provides an exciting instrument for measuring the mechanical properties of single cells. However, current AFM single-cell mechanical measurements are commonly performed on cell lines cultured in vitro which are quite different from the primary cells in the human body. Investigating the mechanical properties of primary cells from clinical environments can help us to better understand cell behaviors. Here, by combining AFM with magnetic beads cell isolation, the viscoelastic properties of human primary B lymphocytes were quantitatively measured. B lymphocytes were isolated from the peripheral blood of healthy volunteers by density gradient centrifugation and CD19 magnetic beads cell isolation. The activity and specificity of the isolated cells were confirmed by fluorescence microscopy. AFM imaging revealed the surface topography and geometric parameters of B lymphocytes. The instantaneous modulus and relaxation time of living B lymphocytes were measured by AFM indenting technique, showing that the instantaneous modulus of human normal B lymphocytes was 2-3 kPa and the relaxation times were 0.03-0.06 s and 0.35-0.55 s. The differences in cellular visocoelastic properties between primary B lymphocytes and cell lines cultured in vitro were analyzed. The study proves the capability of AFM in quantifying the viscoelastic properties of individual specific primary cells from the blood sample of clinical patients, which will improve our understanding of the behaviors of cells in the human body.

Improved detection of Bovine Viral Diarrhea Virus in Bovine lymphoid cell lines using PrimeFlow RNA assay

USDA-ARS?s Scientific Manuscript database

Bovine viral diarrhea virus (BVDV) infections, whether as acute, persistent or contributing to co-infections, result in significant losses for cattle producers. BVDV can be identified by real-time PCR and ELISA, detection and quantification of viral infection at the single cell level is extremely di...

Improved detection of bovine viral diarrhea virus in bovine lymphoid cell lines using PrimeFlow RNA assay

USDA-ARS?s Scientific Manuscript database

Bovine viral diarrhea virus (BVDV) infections, whether as acute, persistent or contributing to co-infections, result in significant losses for cattle producers. BVDV can be identified by real-time PCR and ELISA, detection and quantification of viral infection at the single cell level is extremely di...

Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics.

PubMed

Hosokawa, Masahito; Nishikawa, Yohei; Kogawa, Masato; Takeyama, Haruko

2017-07-12

Massively parallel single-cell genome sequencing is required to further understand genetic diversities in complex biological systems. Whole genome amplification (WGA) is the first step for single-cell sequencing, but its throughput and accuracy are insufficient in conventional reaction platforms. Here, we introduce single droplet multiple displacement amplification (sd-MDA), a method that enables massively parallel amplification of single cell genomes while maintaining sequence accuracy and specificity. Tens of thousands of single cells are compartmentalized in millions of picoliter droplets and then subjected to lysis and WGA by passive droplet fusion in microfluidic channels. Because single cells are isolated in compartments, their genomes are amplified to saturation without contamination. This enables the high-throughput acquisition of contamination-free and cell specific sequence reads from single cells (21,000 single-cells/h), resulting in enhancement of the sequence data quality compared to conventional methods. This method allowed WGA of both single bacterial cells and human cancer cells. The obtained sequencing coverage rivals those of conventional techniques with superior sequence quality. In addition, we also demonstrate de novo assembly of uncultured soil bacteria and obtain draft genomes from single cell sequencing. This sd-MDA is promising for flexible and scalable use in single-cell sequencing.

Low-power laser effects at the single-cell level: a confocal microscopy study

NASA Astrophysics Data System (ADS)

Alexandratou, Eleni; Yova, Dido M.; Atlamazoglou, Vassilis; Handris, Panagiotis; Kletsas, Dimitris; Loukas, Spyros

2000-11-01

Confocal microscopy was used for irradiation and observation of the same area of interest, allowing the imaging of low power laser effects in subcellular components and functions, at the single cell level. Coverslips cultures of human fetal foreskin fibroblasts (HFFF2) were placed in a small incubation chamber for in vivo microscopic observation. Cells were stimulated by the 647 nm line of the Argon- Krypton laser of the confocal microscope (0.1 mW/cm2). Membrane permeability, mitochondrial membrane potential ((delta) Psim), intracellular pHi, calcium alterations and nuclear chromatin accessibility were monitored, at different times after irradiation, using specific fluorescent vital probes. Images were stored to the computer and quantitative evaluation was performed using image- processing software. After irradiation, influx and efflux of the appropriate dyes monitored changes in cell membrane permeability. Laser irradiation caused alkalizatoin of the cytosolic pHi and increase of the mitochondrial membrane potential ((delta) Psim). Temporary global Ca2+ responses were also observed. No such effects were noted in microscopic fields other than the irradiated ones. No toxic effects were observed, during time course of the experiment.

Individual blastomeres of 16- and 32-cell mouse embryos are able to develop into foetuses and mice.

PubMed

Tarkowski, Andrzej K; Suwińska, Aneta; Czołowska, Renata; Ożdżeński, Wacław

2010-12-15

Cell and developmental studies have clarified how, by the time of implantation, the mouse embryo forms three primary cell lineages: epiblast (EPI), primitive endoderm (PE), and trophectoderm (TE). However, it still remains unknown when cells allocated to these three lineages become determined in their developmental fate. To address this question, we studied the developmental potential of single blastomeres derived from 16- and 32-cell stage embryos and supported by carrier, tetraploid blastomeres. We were able to generate singletons, identical twins, triplets, and quadruplets from individual inner and outer cells of 16-cell embryos and, sporadically, foetuses from single cells of 32-cell embryos. The use of embryos constitutively expressing GFP as the donors of single diploid blastomeres enabled us to identify their cell progeny in the constructed 2n↔4n blastocysts. We showed that the descendants of donor blastomeres were able to locate themselves in all three first cell lineages, i.e., epiblast, primitive endoderm, and trophectoderm. In addition, the application of Cdx2 and Gata4 markers for trophectoderm and primitive endoderm, respectively, showed that the expression of these two genes in the descendants of donor blastomeres was either down- or up-regulated, depending on the cell lineage they happened to occupy. Thus, our results demonstrate that up to the early blastocysts stage, the destiny of at least some blastomeres, although they have begun to express markers of different lineage, is still labile. Copyright © 2010 Elsevier Inc. All rights reserved.

Hydrogel microstructure live-cell array for multiplexed analyses of cancer stem cells, tumor heterogeneity and differential drug response at single-element resolution.

PubMed

Afrimzon, E; Botchkina, G; Zurgil, N; Shafran, Y; Sobolev, M; Moshkov, S; Ravid-Hermesh, O; Ojima, I; Deutsch, M

2016-03-21

Specific phenotypic subpopulations of cancer stem cells (CSCs) are responsible for tumor development, production of heterogeneous differentiated tumor mass, metastasis, and resistance to therapies. The development of therapeutic approaches based on targeting rare CSCs has been limited partially due to the lack of appropriate experimental models and measurement approaches. The current study presents new tools and methodologies based on a hydrogel microstructure array (HMA) for identification and multiplex analyses of CSCs. Low-melt agarose integrated with type I collagen, a major component of the extracellular matrix (ECM), was used to form a solid hydrogel array with natural non-adhesive characteristics and high optical quality. The array contained thousands of individual pyramidal shaped, nanoliter-volume micro-chambers (MCs), allowing concomitant generation and measurement of large populations of free-floating CSC spheroids from single cells, each in an individual micro-chamber (MC). The optical live cell platform, based on an imaging plate patterned with HMA, was validated using CSC-enriched prostate and colon cancer cell lines. The HMA methodology and quantitative image analysis at single-element resolution clearly demonstrates several levels of tumor cell heterogeneity, including morphological and phenotypic variability, differences in proliferation capacity and in drug response. Moreover, the system facilitates real-time examination of single stem cell (SC) fate, as well as drug-induced alteration in expression of stemness markers. The technology may be applicable in personalized cancer treatment, including multiplex ex vivo analysis of heterogeneous patient-derived tumor specimens, precise detection and characterization of potentially dangerous cell phenotypes, and for representative evaluation of drug sensitivity of CSCs and other types of tumor cells.

Photothermal confocal multicolor microscopy of nanoparticles and nanodrugs in live cells

PubMed Central

Nedosekin, Dmitry A.; Foster, Stephen; Nima, Zeid A.; Biris, Alexandru S.; Galanzha, Ekaterina I.; Zharov, Vladimir P.

2018-01-01

Growing biomedical applications of non-fluorescent nanoparticles (NPs) for molecular imaging, disease diagnosis, drug delivery, and theranostics require new tools for real-time detection of nanomaterials, drug nano-carriers and NP-drug conjugates (nanodrugs) in complex biological environments without additional labeling. Photothermal (PT) microscopy (PTM) has an enormous potential for absorption-based identification and quantification of non-fluorescent molecules and NPs at a single molecule and 1.4 nm gold NP level. Recently, we have developed confocal PTM providing three-dimensional (3-D) mapping and spectral identification of multiple chromophores and fluorophores in live cells. Here, we summarize recent advances in the application of confocal multicolor PTM for 3-D visualization of single and clustered NPs, alone and in individual cells. In particular, we demonstrate identification of functionalized magnetic and gold-silver NPs, as well as graphene and carbon nanotubes in cancer cells and among blood cells. The potentials to use PTM for super-resolution imaging (down to 50nm), real-time NP tracking, guidance of PT nanotherapy and multiplex cancer markers targeting, as well as analysis of nonlinear PT phenomena and amplification of nanodrug efficacy through NP clustering and nanobubble formation are also discussed. PMID:26133539

Reducing background contributions in fluorescence fluctuation time-traces for single-molecule measurements in solution.

PubMed

Földes-Papp, Zeno; Liao, Shih-Chu Jeff; You, Tiefeng; Barbieri, Beniamino

2009-08-01

We first report on the development of new microscope means that reduce background contributions in fluorescence fluctuation methods: i) excitation shutter, ii) electronic switches, and iii) early and late time-gating. The elements allow for measuring molecules at low analyte concentrations. We first found conditions of early and late time-gating with time-correlated single-photon counting that made the fluorescence signal as bright as possible compared with the fluctuations in the background count rate in a diffraction-limited optical set-up. We measured about a 140-fold increase in the amplitude of autocorrelated fluorescence fluctuations at the lowest analyte concentration of about 15 pM, which gave a signal-to-background advantage of more than two-orders of magnitude. The results of this original article pave the way for single-molecule detection in solution and in live cells without immobilization or hydrodynamic/electrokinetic focusing at longer observation times than are currently available.

Preliminary investigation of single chamber single electrode microbial fuel cell using sewage sludge as a substrate

NASA Astrophysics Data System (ADS)

Sai Chaithanya, M.; Thakur, Somil; Sonu, Kumar; Das, Bhaskar

2017-11-01

A microbial fuel cell (MFC) consists of a cathode and anode; micro-organisms transfer electrons acquired from the degradation of organic matter in the substrate to anode; and thereby to cathode; by using an external circuit to generate electricity. In the present study, a single chamber single electrode microbial fuel cell has been fabricated to generate electricity from the sludge of the sewage treatment plant at two different ambient temperature range of 25 ± 4°C and 32 ± 4°C under aerobic condition. No work has been done yet by using the single electrode in any MFC system; it is hypothesized that single electrode submerged partially in substrate and rest to atmosphere can function as both cathode and anode. The maximum voltage obtained was about 2890 mV after 80 (hrs) at temperature range of 25 ± 4°C, with surface power density of 1108.29 mW/m2. When the ambient temperature was 32 ± 4°C, maximum voltage obtained was 1652 mV after 40 (hrs.) surface power density reduced to 865.57 mW/m2. When amount of substrate was decreased for certain area of electrode at 25 ± 4°C range, electricity generation decreased and it also shortened the time to reach peak voltage. On the other hand, when the ambient temperature was increased to 32 ± 4°C, the maximum potential energy generated was less than that of previous experiment at 25 ± 4°C for the same substrate Also the time to reach peak voltage decreased to 40 hrs. When comparing with other single chamber single electrode MFC, the present model is generating more electricity that any MFC using sewage sludge as substrate except platinum electrode, which is much costlier that electrode used in the present study.