Download or read book Microfluidic Tools for Connecting Single Cell Optical and Gene Expression Phenotype written by Jesse Qiuxu Zhang and published by . This book was released on 2020 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: The single cell is the fundamental unit of biology. Understanding how the identity of individual cells in multicellular organisms contribute to their function remains a key question in Biology. Traditionally, most observations of cells were made through imaging-based techniques. Today, advances in Next Generation Sequencing have led to the widespread adoption of sequencing-based techniques for investigating the genotype and phenotype at single-cell resolution. Microfluidics, including droplet-based microfluidics, have been instrumental in the most successful commercial single-cell genomics platforms.Integrating sequencing and imaging techniques will provide additional information than either of the techniques alone. Both single-cell imaging and genomics techniques measure orthogonal targets, and when combined reveal additional insights into cellular function. However, when performing sequential single-cell assays, there currently exists a tradeoff between throughput and information content. This dissertation will describe progress made towards reducing that gap. I will describe novel microfluidic platforms and techniques and applications involving integrating single-cell sequencing and optical measurements at high throughput. The microfluidics tools that will be discussed in this Dissertation aim to be a platform for performing single-cell multi-parameter and multi-omics techniques that will help further our understanding of cellular identity and how genotype informs phenotype at the single-cell level.

Download or read book Functional Metagenomics Tools and Applications written by Trevor C. Charles and published by Springer. This book was released on 2017-10-09 with total page 256 pages. Available in PDF, EPUB and Kindle. Book excerpt: In this book, the latest tools available for functional metagenomics research are described.This research enables scientists to directly access the genomes from diverse microbial genomes at one time and study these “metagenomes”. Using the modern tools of genome sequencing and cloning, researchers have now been able to harness this astounding metagenomic diversity to understand and exploit the diverse functions of microorganisms. Leading scientists from around the world demonstrate how these approaches have been applied in many different settings, including aquatic and terrestrial habitats, microbiomes, and many more environments. This is a highly informative and carefully presented book, providing microbiologists with a summary of the latest functional metagenomics literature on all specific habitats.

Download or read book Microfluidics for Single Cell Analysis written by Jin-Ming Lin and published by Springer Nature. This book was released on 2019-08-28 with total page 263 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book summarizes the various microfluidic-based approaches for single-cell capture, isolation, manipulation, culture and observation, lysis, and analysis. Single-cell analysis reveals the heterogeneities in morphology, functions, composition, and genetic performance of seemingly identical cells, and advances in single-cell analysis can overcome the difficulties arising due to cell heterogeneity in the diagnostics for a targeted model of disease. This book provides a detailed review of the state-of-the-art techniques presenting the pros and cons of each of these methods. It also offers lessons learned and tips from front-line investigators to help researchers overcome bottlenecks in their own studies. Highlighting a number of techniques, such as microfluidic droplet techniques, combined microfluidics-mass-spectrometry systems, and nanochannel sampling, it describes in detail a new microfluidic chip-based live single-cell extractor (LSCE) developed in the editor’s laboratory, which opens up new avenues to use open microfluidics in single-cell extraction, single-cell mass spectrometric analysis, single-cell adhesion analysis and subcellular operations. Serving as both an elementary introduction and advanced guidebook, this book interests and inspires scholars and students who are currently studying or wish to study microfluidics-based cell analysis methods.

Download or read book Microfluidics in Cell Biology Part B Microfluidics in Single Cells written by and published by Academic Press. This book was released on 2018-08-27 with total page 250 pages. Available in PDF, EPUB and Kindle. Book excerpt: Microfluidics in Cell Biology Part B: Microfluidics in Single Cells, Volume 147, a new volume in the Methods in Cell Biology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. Unique to this updated volume are three sections on microfluidics in various single cell models, including microfludics in micro-organisms, microfluidics for cell culture and cell sorting of mammalian cells, and microfluidics for cell migration. Specific sections in this latest release include Temperature control and drug delivery for cell division cycle control in fission yeast H2O2 stress response in budding yeast, Antibiotic resistance in bacteria, Metabolism in bacteria, Fluidized beds for bacterial sorting and amplification, Microfluidics for cell culture and cell sorting of mammalian cells, Hydrogel microwells, Immune cells migration in complex environments, Neutrophiles migration in health and disease, Cell guidance by physical cues, Stable gradients in gels of extracellular matrix for cancer cell migration, and more. Contains contributions from experts in the field from across the world Covers a wide array of topics on both mitosis and meiosis Includes relevant, analysis based topics

Download or read book Unravelling Single Cell Genomics written by Nathalie Bontoux and published by Royal Society of Chemistry. This book was released on 2010-10-18 with total page 333 pages. Available in PDF, EPUB and Kindle. Book excerpt: This unique introduction to the growing field of microfluidics applied to genomics provides an overview of the latest technologies and emphasizes its potential in answering important biological questions. Written by a physicist and a biologist, it offers a more comprehensive view than the previous literature. The book starts with key ideas in molecular biology, developmental biology and microtechnology before going on to cover the specifics of single cell analysis and microfluidic devices for single cell molecular analysis. Review chapters discuss the state-of-the art and will prove invaluable to all those planning to develop microdevices for molecular analysis of single cells. Methods allowing complete analysis of gene expression in the single cell are stressed - as opposed the more commonly used techniques that allow analysis of only a few genes at a time. As pioneers in the field, the authors understand how critical it is for a physicist to understand the biological issues and questions related to single cell analysis, as well for biologists to understand what microfluidics is all about. Aimed predominantly at graduate students, this book will also be of significant interest to scientists working in or affiliated with this field.

Download or read book Microfluidic Technology for High throughput Single Cell Gene Expression Analysis written by Adam White and published by . This book was released on 2010 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Micro Nanofluidic Devices for Single Cell Analysis written by Fan-Gang Tseng and published by MDPI. This book was released on 2018-10-04 with total page 167 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book is a printed edition of the Special Issue "Micro/Nanofluidic Devices for Single Cell Analysis" that was published in Micromachines

Download or read book Microfluidic Methods for Molecular Biology written by Chang Lu and published by Springer. This book was released on 2016-05-14 with total page 382 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book covers the state-of-the-art research on molecular biology assays and molecular techniques enabled or enhanced by microfluidic platforms. Topics covered include microfluidic methods for cellular separations and single cell studies, droplet-based approaches to study protein expression and forensics, and microfluidic in situ hybridization for RNA analysis. Key molecular biology studies using model organisms are reviewed in detail. This is an ideal book for students and researchers in the microfluidics and molecular biology fields as well as engineers working in the biotechnology industry. This book also: Reviews exhaustively the latest techniques for single-cell genetic, epigenetic, metabolomic, and proteomic analysis Illustrates microfluidic approaches for inverse metabolic engineering, as well as analysis of circulating exosomes Broadens readers’ understanding of microfluidics convection-based PCR technology, microfluidic RNA-seq, and microfluidics for robust mobile diagnostics

Download or read book A Microfluidic Platform for the Genome wide Analysis of Electrical Phenotype written by Michael David Vahey and published by . This book was released on 2010 with total page 143 pages. Available in PDF, EPUB and Kindle. Book excerpt: This thesis presents the development of a new microfluidic method for separating and characterizing cells based upon differences in their electrical properties. The objective of this work is to obtain a genome-wide mapping of genotype to electrical phenotype in the budding yeast Saccharomyces cerevisiae. Towards this end, we present: (1) the development of a novel equilibrium gradient separation method, called isodielectric separation (IDS); (2) the development of physical theories describing how interactions between particles effect microscale separations; and (3) the application of IDS to a screen for electrical phenotypes in the yeast deletion library. Despite the variety of technologies available for cell sorting, a relative lack of intrinsic separation methods - those which separate cells according to their natural, unmodified characteristics - persists. To address this need, we have developed isodielectric separation. IDS separates cells according to differences in their intrinsic electrical properties. Using dielectrophoresis in a medium with spatially varying electrical conductivity, IDS drives cells to the locations where their polarization charges vanish, spatially resolving cells with different electrical properties. Our implementation of IDS offers label-free, continuous-flow separation, and is capable of resolving graded differences in electrical properties. Additionally, we demonstrate the ability to extract quantitative information from samples during separation, establishing IDS as an analytical technique as well as a preparative one. Any platform for performing genetic screens must have high throughput. Although satisfying this requirement would be greatly facilitated by using high cell densities, physical interactions between cells under these conditions can affect the performance of devices used for screening. Although pervasive, interactions between cells or particles are challenging to describe quantitatively, especially in the confined environments typical of microfluidic devices. By studying the effects of electrostatic and hydrodynamic interactions between particles in a microfluidic device, we have found that ensembles of interacting particles exhibit emergent behaviors that we are able to predict through numerical simulations and a simple analytic model based on hydrodynamic coupling. Applying these theoretical tools to IDS and other microfluidic separation methods has provided insight into how particle interactions can profoundly influence separation performance in counterintuitive ways. Having demonstrated the performance metrics necessary for a genetic screen, we apply IDS to the genome-wide analysis of electrical properties in the budding yeast S. cerevisiae. Although others have studied changes in electrical properties induced by drastic changes in gene expression (e.g. in differentiation) or by specific mutations in a small number of genes, a systematic and comprehensive analysis of the relationship between genotype and electrical phenotype has yet to be performed. Using IDS, we have screened the -5000 strains in the yeast deletion collection for altered electrical phenotype. This work has identified a number of genes associated with distinct electrical properties, and, by analyzing known interactions and correlations between these genes, we have identified pathways and morphologies that appear to be primary determinants of electrical phenotype.

Download or read book Microfluidics for Cells and Other Organisms written by Danny van Noort and published by MDPI. This book was released on 2019-10-23 with total page 200 pages. Available in PDF, EPUB and Kindle. Book excerpt: Microfluidics-based devices play an important role in creating realistic microenvironments in which cell cultures can thrive. They can, for example, be used to monitor drug toxicity and perform medical diagnostics, and be in a static-, perfusion- or droplet-based device. They can also be used to study cell-cell, cell-matrix or cell-surface interactions. Cells can be either single cells, 3D cell cultures or co-cultures. Other organisms could include bacteria, zebra fish embryo, C. elegans, to name a few.

Download or read book Optimization of Trustworthy Biomolecular Quantitative Analysis Using Cyber Physical Microfluidic Platforms written by Mohamed Ibrahim and published by CRC Press. This book was released on 2020-05-31 with total page 335 pages. Available in PDF, EPUB and Kindle. Book excerpt: A microfluidic biochip is an engineered fluidic device that controls the flow of analytes, thereby enabling a variety of useful applications. According to recent studies, the fields that are best set to benefit from the microfluidics technology, also known as lab-on-chip technology, include forensic identification, clinical chemistry, point-of-care (PoC) diagnostics, and drug discovery. The growth in such fields has significantly amplified the impact of microfluidics technology, whose market value is forecast to grow from $4 billion in 2017 to $13.2 billion by 2023. The rapid evolution of lab-on-chip technologies opens up opportunities for new biological or chemical science areas that can be directly facilitated by sensor-based microfluidics control. For example, the digital microfluidics-based ePlex system from GenMarkDx enables automated disease diagnosis and can bring syndromic testing near patients everywhere. However, as the applications of molecular biology grow, the adoption of microfluidics in many applications has not grown at the same pace, despite the concerted effort of microfluidic systems engineers. Recent studies suggest that state-of-the-art design techniques for microfluidics have two major drawbacks that need to be addressed appropriately: (1) current lab-on-chip systems were only optimized as auxiliary components and are only suitable for sample-limited analyses; therefore, their capabilities may not cope with the requirements of contemporary molecular biology applications; (2) the integrity of these automated lab-on-chip systems and their biochemical operations are still an open question since no protection schemes were developed against adversarial contamination or result-manipulation attacks. Optimization of Trustworthy Biomolecular Quantitative Analysis Using Cyber-Physical Microfluidic Platforms provides solutions to these challenges by introducing a new design flow based on the realistic modeling of contemporary molecular biology protocols. It also presents a microfluidic security flow that provides a high-level of confidence in the integrity of such protocols. In summary, this book creates a new research field as it bridges the technical skills gap between microfluidic systems and molecular biology protocols but it is viewed from the perspective of an electronic/systems engineer.

Download or read book Integrated Microfluidic Device for Single cell High Throughput Screening in Dynamic Gene Expression Analysis written by Lawrence Kwan Yeung Hui and published by . This book was released on 2008 with total page 60 pages. Available in PDF, EPUB and Kindle. Book excerpt: Over the past decade, interest in microfluidics has surged as applications have trended towards novel biological assays. Specifically, the ability of microfluidics to parallelize cellular studies through array-based chip designs has attracted researchers interested in investigating cellular function under a wide variety of environmental conditions. The capability of microfluidic devices to control microenvironment conditions and induce dynamic perturbation to cellular systems makes microfluidics (or "lab-on-a-chip") an attractive platform to study gene expression dynamics. In this project, the functionality of microfluidic technology is exploited to design and construct a device for isolation and observation of cells in high throughput. The integration of a concentration gradient with homogenous medium within each chamber was designed specifically to investigate gene regulation in Saccharomyces cerevisiae under various concentrations of chemical inducers. These devices were designed to sustain cells for extended periods of time with high temporal resolution to study dynamic gene expression in single cells. The device builds on previous studies by probing up to eight distinct cell cultures in parallel. The microfluidic platform was then used to study yeast cells at various levels of inducer perturbations. Further experimentation revealed the utility of a parallel gradient by producing an induction curve of the yeast response. Such high-throughput designs will prove essential to yeast systems biology research as it strives to understand the complex regulatory interactions that dictate cell function by probing vast regions of parameter space.

Download or read book Microfluidics for Synthetic Biology written by Ivan Alexandrovich Razinkov and published by . This book was released on 2013 with total page 185 pages. Available in PDF, EPUB and Kindle. Book excerpt: Bottom-up approach in understanding and developing gene networks calls for study of cells on a single cell level. Microfluidic technology provides such necessary tools to examine single cells not only in static but even more importantly in dynamic environments. Here we present studies that focus on examining model organisms of Escherichia coli and Saccharomyces cerevisiae on single cell and colony level. First, we developed a microfluidic device for studying single cell response of Galactose network of S. cerevisiae in fluctuating carbon source environment. As part of the study we have developed a novel off-chip technology that allows for fast and dynamic control of extracellular environment. In addition, this microfluidic device allows for 8 independent single cell level experiments to be run simultaneously. Next, this technology was adapted to study protein expression on single-protein level in E. coli. Furthermore, we developed a microfluidic device to study and characterize a colony-coupled synthetic oscillator in E. coli. To study the diffusion characteristics of the coupling agent the scale of the devices was increased from nominal 100,000 cells to include over 50 million cells. Lastly, we combined the ability to generate a dynamic environment with a large-scale device to study long-term population dynamics of two competing S. cerevisiae strains.

Download or read book Microfluidics for Investigating Single cell Biodynamics written by Scott Warren Cookson and published by . This book was released on 2008 with total page 124 pages. Available in PDF, EPUB and Kindle. Book excerpt: Progress in synthetic biology requires the development of novel techniques for investigating long-term dynamics in single cells. Here, we demonstrate the utility of microfluidics for investigating single-cell biodynamics within tightly-controlled environments in the model organisms Saccharomyces cerevisiae and Escherichia coli. First, we develop a microfluidic chemostat for monitoring single-cell gene expression within large populations of S. cerevisiae over many cellular generations. We overcome typical difficulties in tracking individual cells throughout long sequences of time-lapse microscopy images by constraining colony growth to a monolayer. Second, we construct a variant of this device to elucidate a new mechanism of cellular ordering within dense E. coli populations. By comparing colony growth and alignment inside a long and narrow microfluidic channel with continuum models and discrete element simulations, we conclude that the observed dynamic transition from an isotropic disordered phase to a nematic ordered phase is caused by biomechanical interactions arising from cellular growth and division. Finally, we use microfluidics and time-lapse microscopy to characterize an engineered genetic oscillator in E. coli. We find the circuit to exhibit fast and robust oscillatory periods that can be tuned by altering inducer levels, temperature, and the growth medium. Together, these studies illustrate the role of novel measurement techniques in advancing the goals of synthetic biology. Specifically, the ability to generate single-cell expression profiles for a large number of cells is essential to understanding the roles of regulatory motifs within native and synthetic gene networks. Through such an understanding, we aim to develop an engineering-based approach to building gene-regulatory circuits, where design specifications generated from computational modeling drive the construction of regulatory networks with desired properties.

Download or read book Microfluidic Fractionation and Analysis of Cytoplasmic Versus Nuclear Acids in Single Cells written by Denitsa Milanova and published by . This book was released on 2015 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Single-cell gene expression studies have matured and are now widely used to uncover cell-to-cell variability, transcript processing and inhomogeneous response to external stimuli. Despite great progress, significant challenges remain including understanding fundamental questions of human genome transcription, gene expression ranges, localization, and processing destinies of RNAs. Eukaryotic cells make different types of primary and processed transcripts, which are either exclusively found in particular sub-cellular compartments or dispersed throughout the whole cell. These sub-cellular localizations of RNAs are weakly understood at a single-cell level, and are important for fully realizing gene functions. Understanding sub-cellular transcript localization is particularly important for studying the process of splicing, a pathway during which introns of pre-messenger RNA's are excised and exons are stitched together to form mature mRNA transcript. We here introduce a single-cell-isotachophoresis (sc-ITP) method to analyze co-transcriptional and alternative splicing in reference lymphoblastoid cell line (LCL-Snyder), and two sub-lines of CML cells (K562-ATCC and K562-ENCODE). The method is unique in that we physically separate the contents of cell nucleus from those of the cytoplasm and analyze independently. It allows for rapid, electric-field-based selective lysis of cytoplasmic membrane (leaving nucleus intact); and then purification and simultaneous fractionation of total RNA in cytosol (cyt-RNA) and total RNA in the nucleus (nuc-RNA) from single cells with no intra-compartment cross-contamination. We then use our system and state-of-the-art NGS technologies to perform single-cell nuclear and cytoplasmic RNA-seq to study fundamental questions of human genome transcription, differential gene expression, localization, and processing destinies of RNAs for various cell types, disease, and differentiation states. We evaluate the distribution of whole transcriptome gene features and gene expression of precursor and processed transcripts and find that there are considerable differences in expression levels across sub-cellular compartments and among individual cells. We then evaluate cell-to-cell variability of the highly expressed GAPDH housekeeping gene in alternative splicing between the lymphoblastoid and leukaemia cells using sequencing-specific probes and RT-qPCR. The data suggest evidence of significant distinction in splicing patterns. By analyzing nuclear and cytoplasmic compartments of a single cell individually at a whole transcriptome level, we achieve an unprecedented precision in splicing quantification. Together, our results describe an experimental method for single-cell fractionation and an analytical tool for gene and isoform expression analysis in rare cell types, cell differentiation and disease states.

Download or read book Microfluidics for Single cell Analysis written by and published by . This book was released on 2019 with total page 263 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book summarizes the various microfluidic-based approaches for single-cell capture, isolation, manipulation, culture and observation, lysis, and analysis. Single-cell analysis reveals the heterogeneities in morphology, functions, composition, and genetic performance of seemingly identical cells, and advances in single-cell analysis can overcome the difficulties arising due to cell heterogeneity in the diagnostics for a targeted model of disease. This book provides a detailed review of the state-of-the-art techniques presenting the pros and cons of each of these methods. It also offers lessons learned and tips from front-line investigators to help researchers overcome bottlenecks in their own studies. Highlighting a number of techniques, such as microfluidic droplet techniques, combined microfluidics-mass-spectrometry systems, and nanochannel sampling, it describes in detail a new microfluidic chip-based live single-cell extractor (LSCE) developed in the editor's laboratory, which opens up new avenues to use open microfluidics in single-cell extraction, single-cell mass spectrometric analysis, single-cell adhesion analysis and subcellular operations. Serving as both an elementary introduction and advanced guidebook, this book interests and inspires scholars and students who are currently studying or wish to study microfluidics-based cell analysis methods.

Download or read book Droplet based Microfluidics for the Genotype phenotype Mapping of Model Enzymes written by Dany Chauvin and published by . This book was released on 2017 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: The question of how sequence encodes proteins' function is essential to understand molecular evolution but still remains elusive.Droplet-based microfluidics allows to use micro-metric droplets as reaction vessels to separately assay enzyme variants at the kHz frequency. It also provides an elegant solution to couple the genotype with the product of the catalytic activity of enzymes. Sorting droplets on demand and sequencing their content enables to map the genotype of millions of enzyme variants to their phenotype in a single experiment.First, I developed a cell-free microfluidic work-flow to carry out genotype-phenotype mapping of Streptomyces griseus aminopeptidase (SGAP). Single enzyme variant genes are encapsulated and amplified in droplets, expressed, and assayed against a fluorogenic substrate. Incompatibilities between gene amplification, expression and assay reactions, constrain to execute each one of those steps successively and to dilute the product of each reaction by droplet electro-coalescence. I show that a work-flow in which (i) genes are encapsulated and amplified into 0.2 pL droplets, (ii) expressed using cell-free expression reagents in 2 pL droplets and (iii) assayed with a fluorogenic substrate in 20 pL droplets, allows to measure SGAP variants activity with high contrast. To optimize the SGAP droplet assay, I also developed in collaboration with Dr. Johan Fenneteau (Laboratory of Organic Chemistry, ESPCI Paristech), a hydrophilic rhodamine based substrate, characterized by limited exchange of the released fluorophore between droplets.Second, I developed an in vivo microfluidic work-flow on Ratus norvegicus trypsin (rat trypsin), in which Bacillus subtilis secretion abilities are used to simplify the microfluidic work-flow. Single B. subtilis cells are encapsulated in 20 pL droplets where they secrete trypsin variants as fusion proteins with a fluorescent expression-level reporter. The variants are assayed by droplet electro-coalescence with 2 pL droplets containing a trypsin fluorogenic substrate. Trypsin variants catalytic efficiency can be directly measured in droplets, by normalizing the total trypsin activity by the expression-level reporter fluorescence. This is the first time a high-throughput protein assay work-flow gives the opportunity to directly measure the catalytic efficiency of enzyme variants at the kHz frequency. A method to carry out saturated mutagenesis on the rat trypsin gene was also developed. Together with deep sequencing, the developed experimental work-flow will allow to perform the first quantitative genotype-phenotype mapping of all single point mutants of the rat trypsin protein.