| 1. |
- Khorshidi, Mohammad Ali, 1981-
(författare)
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Live Single Cell Imaging and Analysis Using Microfluidic Devices
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Today many cell biological techniques study large cell populations where an average estimate of individual cells’ behavior is observed. On the other hand, single cell analysis is required for studying functional heterogeneities between cells within populations. This thesis presents work that combines the use of microfluidic devices, optical microscopy and automated image analysis to design various cell biological assays with single cell resolution including cell proliferation, clonal expansion, cell migration, cell-cell interaction and cell viability tracking. In fact, automated high throughput single cell techniques enable new studies in cell biology which are not possible with conventional techniques.In order to automatically track dynamic behavior of single cells, we developed a microwell based device as well as a droplet microfluidic platform. These high throughput microfluidic assays allow automated time-lapse imaging of encapsulated single cells in micro droplets or confined cells inside microwells. Algorithms for automatic quantification of cells in individual microwells and micro droplets are developed and used for the analysis of cell viability and clonal expansion. The automatic counting protocols include several image analysis steps, e.g. segmentation, feature extraction and classification. The automatic quantification results were evaluated by comparing with manual counting and revealed a high success rate. In combination these automatic cell counting protocols and our microfluidic platforms can provide statistical information to better understand behavior of cells at the individual level under various conditions or treatments in vitro exemplified by the analysis of function and regulation of immune cells. Thus, together these tools can be used for developing new cellular imaging assays with resolution at the single cell level.To automatically characterize transient migration behavior of natural killer (NK) cells compartmentalized in microwells, we developed a method for single cell tracking. Time-lapse imaging showed that the NK cells often exhibited periods of high motility, interrupted with periods of slow migration or complete arrest. These transient migration arrest periods (TMAPs) often overlapped with periods of conjugations between NK cells and target cells. Such conjugation periods sometimes led to cell-mediated killing of target cells. Analysis of cytotoxic response of NK cells revealed that a small sub-class of NK cells called serial killers was able to kill several target cells. In order to determine a starting time point for cell-cell interaction, a novel technique based on ultrasound was developed to aggregate NK and target cells into the center of the microwells. Therefore, these assays can be used to automatically and rapidly assess functional and migration behavior of cells to detect differences between health and disease or the influence of drugs.The work presented in this thesis gives good examples of how microfluidic devices combined with automated imaging and image analysis can be helpful to address cell biological questions where single cell resolution is necessary.
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| 2. |
- Periyannan Rajeswari, Prem Kumar
(författare)
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Droplet microfluidics for single cell and nucleic acid analysis
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Droplet microfluidics is an emerging technology for analysis of single cells and biomolecules at high throughput. The controlled encapsulation of particles along with the surrounding microenvironment in discrete droplets, which acts as miniaturized reaction vessels, allows millions of particles to be screened in parallel. By utilizing the unit operations developed to generate, manipulate and analyze droplets, this technology platform has been used to miniaturize a wide range of complex biological assays including, but not limited to, directed evolution, rare cell detection, single cell transcriptomics, rare mutation detection and drug screening.The aim of this thesis is to develop droplet microfluidics based methods for analysis of single cells and nucleic acids. In Paper I, a method for time-series analysis of mammalian cells, using automated fluorescence microscopy and image analysis technique is presented. The cell-containing droplets were trapped on-chip and imaged continuously to assess the viability of hundreds of isolated individual cells over time. This method can be used for studying the dynamic behavior of cells. In Paper II, the influence of droplet size on cell division and viability of mammalian cell factories during cultivation in droplets is presented. The ability to achieve continuous cell division in droplets will enable development of mammalian cell factory screening assays in droplets. In Paper III, a workflow for detecting the outcome of droplet PCR assay using fluorescently color-coded beads is presented. This workflow was used to detect the presence of DNA biomarkers associated with poultry pathogens in a sample. The use of color-coded detection beads will help to improve the scalability of the detection panel, to detect multiple targets in a sample. In Paper IV, a novel unit operation for label-free enrichment of particles in droplets using acoustophoresis is presented. This technique will be useful for developing droplet-based assays that require label-free enrichment of cells/particles and removal of droplet content. In general, droplet microfluidics has proven to be a versatile tool for biological analysis. In the years to come, droplet microfluidics could potentially be used to improve clinical diagnostics and bio-based production processes.
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| 3. |
- Gantelius, Jesper, 1980-
(författare)
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Novel diagnostic microarray assay formats towards comprehensive on-site analysis
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Advances in molecular methods for analyzing DNA, RNA and proteins in humans as well as in other animals, plants, fungi, bacteria or viruses have greatly increased the resolution with which we can study life’s complexity and dynamics on earth. While genomic, transcriptomic and proteomic laboratory tools for molecular diagnosis of disease are rapidly becoming more comprehensive, the access to such advanced yet often expensive and centralized procedures is limited. There is a great need for rapid and comprehensive diagnostic methods in low-resource settings or contexts where a person can not or will not go to a hospital or medical laboratory, yet where a clinical analysis is urgent. In this thesis, results from development and characterization of novel technologies for DNA and protein microarray analysis are presented. Emphasis is on methods that could provide rapid, cost-effective and portable analysis with convenient readout and retained diagnostic accuracy. The first study presents a magnetic bead-based approach for DNA microarray analysis for a rapid visual detection of single nucleotide polymorphisms. In the second work, magnetic beads were used as detection reagents for rapid differential detection of presence of pestiviral family members using a DNA oligonucleotide microarray with read-out by means of a tabletop scanner or a digital camera. In paper three, autoimmune responses from human sera were detected on a protein autoantigen microarray, again by means of magnetic bead analysis. Here, special emphasis was made in comprehensively comparing the performance of the magnetic bead detection to common fluorescence-based detection. In the fourth study, an immunochromatographic lateral flow protein microarray assay is presented for application in the classification of contagious pleuropneumonia from bovine serum samples. The analysis could be performed within 10 minutes using a table top scanner, and the performance of the assay was shown to be comparable to that of a cocktail ELISA. In the fifth paper, the lateral flow microarray framework is investigated in further detail by means of experiments and numerical simulation. It was found that downstream effects play an important role, and the results further suggest that the downstream binding profiles may find use in simple affinity evaluation.
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| 4. |
- Lindström, Sara, 1980-
(författare)
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Microwell devices for single-cell analyses
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Powerful tools for detailed cellular studies are emerging, increasing the knowledge ofthe ultimate target of all drugs: the living cell. Today, cells are commonly analyzed inensembles, i.e. thousands of cells per sample, yielding results on the average responseof the cells. However, cellular heterogeneity implies the importance of studying howindividual cells respond, one by one, in order to learn more about drug targeting andcellular behavior. In vitro assays offering low volume sampling and rapid analysis in ahigh-throughput manner are of great interest in a wide range of single-cellapplications. This work presents a microwell device in silicon and glass, developed using standardmicrofabrication techniques. The chip was designed to allow flow-cytometric cellsorting, a controlled way of analyzing and sorting individual cells for dynamic cultureand clone formation, previously shown in larger multiwell plates only. Dependent onthe application, minor modifications to the original device were made resulting in agroup of microwell devices suitable for various applications. Leukemic cancer cellswere analyzed with regard to their clonogenic properties and a method forinvestigation of drug response of critical importance to predict long-term clinicaloutcome, is presented. Stem cells from human and mouse were maintainedpluripotent in a screening assay, also shown useful in studies on neural differentiation.For integrated liquid handling, a fluidic system was integrated onto the chip fordirected and controlled addition of reagents in various cell-based assays. The chip wasproduced in a slide format and used as an imaging tool for low-volume sampling withthe ability to run many samples in parallel, demonstrated in a protein-binding assay fora novel bispecific affinity protein. Moving from cells and proteins into geneticanalysis, a method for screening genes from clones in a rapid manner was shown bygene amplification and mutation analysis in individual wells. In summary, a microwelldevice with associated methods were developed and applied in a range of biologicalinvestigations, particularly interesting from a cell-heterogeneity perspective.
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| 5. |
- Weibull, Emilie, 1982-
(författare)
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Miniaturised Microwell-based Cell Assays
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cell heterogeneity in genetically identical cell populations is becoming a well-known and important phenomenon in cell biology. Current methods commonly utilise population-based analysis founded on averaged result. Hence there is a need for high-throughput cell assays on the single-cell level. By using miniaturised devices it is possible to enhance spatial and temporal control of the individual cells, increase the potential throughput and minimise the needed sample and reagent volume while enabling a wide range of biological applications.This thesis is based on the results generated with a miniaturised microwell slide for cell assays. The microwell slide’s high-throughput compartmentalised configuration enables several hundred isolated experiments to be run simultaneously. The bottom of the wells is made out of a thin glass slide, which supports high-resolution imaging. The slide has a standardised format and its’ compatibility with conventional instruments is used extensively throughout the thesis. The presented papers all contribute to the development of the microwell slide by adding technical value or increasing the number of potential applications. For example, the slide was success-fully implemented as a chip-to-world output format for single microfluidic droplets in Paper I, by interfacing two miniaturised systems with fluorescence-activated cell sorting. In Paper II and III, microfluidic channels were integrated to increase spatial and temporal control of the added samples and reagents. In Paper II an automated stepwise concentration gradient generator was developed delivering a drug gradient to adherent mammalian cells in designated wells. In Paper III fluidic-imposed shear stress on endothelial cells was studied. In Paper IV, the slide was functionalised by coating the surfaces of the wells with several antibiotics at a defined concentration range. The coated slide was used for multiplex antibiotic susceptibility testing of bacterial pathogens, using an algorithm-based identification of the point defining lag to exponential phase transition. It successfully determined the pathogens susceptibility profile in 3-5 hours. Finally, in Paper V, a method to retrieve bacteria colonies with a desired phenotype from the wells for downstream genetic analysis was developed. In summary, the presented work has furthered the development of miniaturised high-throughput tools for various cell heterogeneity assays.
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| 6. |
- Björk, Sara, 1990-
(författare)
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Droplet microfluidics for screening and sorting of microbial cell factories
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cell factories are cells that have been engineered to produce a compound of interest, ranging from biopharmaceuticals to biofuels. With advances in metabolic engineering, the number of cell factory variants to evaluate has increased dramatically, necessitating screening methods with increased throughput. Microfluidic droplets, which can be generated, manipulated and interrogated at very high throughput, are isolated reaction vessels at the single cell scale. Compartmentalization maintains the genotype-phenotype link, making droplet microfluidics suitable for screening of extracellular traits such as secreted products and for screening of microcolonies originating from single cells. In Paper I, we investigated the impact of droplet microfluidic incubation formats on cell culture conditions and found that syringe and semi open incubation resulted in different metabolic profiles. Controlling culture conditions is key to cell factory screening, as product formation is influenced by the state of the cell. In Paper II and III, we used droplet microfluidics to perform screening campaigns of interference based cell factory variant libraries. In Paper II, two S. cerevisiae RNAi libraries were screened based on amylase secretion, and from the sorted fraction genes linked to improved protein secretion could be identified. In paper III, we screened a Synecosystis sp. CRISPRi library based on lactate secretion. The library was sorted at different time points after induction, followed by sequencing to reveal genes enriched by droplet sorting. In Paper IV, we developed a droplet microcolony-based assay for screening intracellular triacylglycerol (TAG) in S. cerevisiae, and showed improved strain separation compared to flow cytometry in a hypothetical sorting scenario. By screening microcolonies compartmentalized in droplets, we combine the throughput of single cell screening methods with the reduced impact of cell-to-cell noise in cell ensemble analysis.
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| 7. |
- Jönsson, Håkan, 1979-
(författare)
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Droplet microfluidics for high throughput biological analysis
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Many areas of biological research increasingly perform large-scale analyses. In genomics the entire gene repertoire of an organism is analyzed. Proteomics attempts to understand the function and expression patterns of all proteins in a cell or organism. Cell biologists study large numbers of single cells to understand the heterogeneity of cell populations. In biotechnology and synthetic biology researchers search for new functional biomolecules in large libraries of biomolecular diversity e.g. for uses in medicine or bioprocessing. More and more all of these fields employ high throughput methods to achieve the scale of analysis necessary. Miniaturization and parallelization provide routes towards high throughput analysis, which have proven successful for microelectronics as well as for DNA sequencing. For the analysis of cells and biomolecules, native to an aqueous environment, miniaturization and parallelization hinges on the handling and parallel processing of very small amounts of water. Droplet microfluidics utilizes stable picoliter (water) droplets contained in inert fluorinated oils as compartments in which to isolate and analyze cells, molecules or reactions. These droplets can be manipulated, detected and analyzed at rates of thousands per second in microfluidic modules combining top-down microscale fabrication with the self-assembly of droplets of exact size. The studies constituting this thesis involve new droplet based biomolecular and single cell assays, manipulation techniques and device fabrication methods to extend the capabilities of droplet microfluidics for high throughput biological analysis. The first paper in the thesis describes a novel analysis method for studying the low abundant biomarkers present on the surface individual cells at resolutions not available by flow cytometry, the current gold standard of single cell analysis. The use of a fluorescent optical dye code enabled the analysis of several single cell samples concurrently, improving throughput. Further a deterministic lateral displacement module, providing passive separation of droplets by size in a microfluidic circuit at more than twice higher rates than previously achievable was demonstrated. Using this module, droplets were separated for cell occupancy based on a cell induced droplet size transformation, which couples a biological property of the droplet contents to a physical property of the droplet. This effect, which enables passive separation of at high throughput, indicates a potential novel assay format for clone selection. One important feature of droplets for encapsulated single cell analysis is retention of secreted molecules providing a genotype-phenotype link. With the objective of detecting antibody molecules secreted by hybridoma for selection, Paper III demonstrates the adaption of a homogeneous fluorescence polarization based, “mix-incubate-read”, assay for antibody detection. In the final paper of the thesis the development of inexpensive and robust optical filters monolithically integrated in the microfluidic chip is reported. These defined filters enable integration of multiple optical filters in a polymer microfluidic device. Overall, droplet microfluidics combines techniques for handling and manipulating millions of discrete biocompatible picoliter compartments per hour with dedicated assays for biomolecule and single cell analysis. The scale of analysis that this enables is certain to impact life science research.
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| 8. |
- Lama, Lara, 1990-
(författare)
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Novel methods for improving rapid paper-based protein assays with gold nanoparticle detection
- 2017
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis describes methods for improving sensitivity in rapid singleplex and multiplex microarray assays. The assays utilize the optical characteristics of colloidal gold nanoparticles for the colorimetric detection of proteins.Multiplexed detection in sandwich immunoassays is limited by cross-reactivity between different detection antibodies. The cross-reactivity between antibodies can contribute to increased background noise - decreasing the Limit-of-Detection of the assay - or generate false positive signals. Paper I shows improved assay sensitivity in a multiplexed vertical flow assay by the application of ultrasonic energy to the gold nanoparticles functionalized with detection antibodies. The ultrasonication of the antibody conjugated gold nanoparticles resulted in a 10 000 fold increase in sensitivity in a 3-plex assay. COMSOL Multiphysics was used to simulate the acoustical energy of the probe used in Paper I for obtaining an indication of the size and direction of the forces acting upon the functionalized gold nanoparticles.In Paper II, it was studied if different gold nanoparticle conjugation methods and colorimetric signal enhancement of the gold nanoparticle conjugates could influence the sensitivity of a paper-based lateral flow microarray assay, targeting cardiac troponin T for the rapid diagnostics of acute myocardial infarction.Ultrasonication and signal enhancement of the detection gold nanoparticles has the potential of improving the sensitivity of paper based assays and expanding their potential future applications.
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| 9. |
- Sjöström, Staffan, 1984-
(författare)
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Droplet microfluidics for directed evolution of biocatalysts
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Biocatalysts, biologically derived catalysts, are of great importance for a wide range of industrial applications. They are used in the production of for example foods, pharmaceuticals and biofuels. Improving biocatalysts commonly relies on directed evolution, i.e. mutagenesis to form diverse variants followed by functional screening in an iterative fashion.Droplet microfluidics is an emerging technology that can be applied for high throughput screening. A key feature of droplet microfluidics is the ability to encapsulate discrete objects, such as single cells, in picoliter-sized droplets at rates of over 1000 cells per second. Each droplet serves as a reaction vessel, analogous to a microwell, where a single clone can be screened.In this thesis, droplet microfluidics is employed for directed evolution of biocatalysts. In paper I, a multiplexed droplet microfluidic method for characterization of enzyme variants is presented and validated by measuring the kinetics of β-galactosidase inhibited by IPTG. In paper II-III, a method for directed evolution of cells with improved production of industrially important enzymes is presented. Two rounds of directed evolution yielded improved strains. The strains had up to 6 times increased enzyme expression levels and whole-genome sequencing revealed 300 mutations, many of which mapped to the protein secretory pathway. In Paper IV, a method for directed evolution of enzyme variants under conditions lethal to host cells is developed. The method is used to screen for α-amylase variants with improved activity or stability at pH4. In Paper V, a method to screen cyanobacteria cell factories is developed and we show that the method can enrich for a strain with high production of L-lactate. In Paper VI, the metabolism of yeast cells encapsulated in microfluidic droplets is studied and found to depend on the choice of emulsion incubation device.Taken together, droplet microfluidics is a promising technology for directed evolution of biocatalysts with the potential to vastly increase throughput and cut costs. The technology could pave the way for process customized biocatalysts and help replace polluting processes with sustainable green chemistry.
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