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Konishi, Yoshinobu. "Live-cell FRET imaging reveals a role of extracellular signal-regulated kinase activity dynamics in thymocyte motility." Kyoto University, 2019. http://hdl.handle.net/2433/242374.
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Hung, Yin Pun. "Single Cell Imaging of Metabolism with Fluorescent Biosensors." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10147.
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Cells utilize various signal transduction networks to regulate metabolism. Nevertheless, a quantitative understanding of the relationship between growth factor signaling and metabolic state at the single cell level has been lacking. The signal transduction and metabolic states could vary widely among individual cells. However, such cell-to-cell variation might be masked by the bulk measurements obtained from conventional biochemical methods. To assess the spatiotemporal dynamics of metabolism in individual intact cells, we developed genetically encoded biosensors based on fluorescent proteins. As a key redox cofactor in metabolism, NADH has been implicated in the Warburg effect, the abnormal metabolism of glucose that is a hallmark of cancer cells. To date, however, sensitive and specific detection of NADH in the cytosol of individual live cells has been difficult. We engineered a fluorescent biosensor of NADH by combining a circularly permuted green fluorescent protein variant with a bacterial NADH-binding protein Rex. The optimized biosensor Peredox reports cytosolic \(NADH:NAD^+\) ratios in individual live cells and can be calibrated with exogenous lactate and pyruvate. Notably pH resistant, this biosensor can be used in several cultured and primary cell types and in a high-content imaging format. We then examined the single cell dynamics of glycolysis and energy-sensing signaling pathways using Peredox and other fluorescent biosensors: AMPKAR, a sensor of the AMPK activity; and FOXO3-FP, a fluorescently-tagged protein domain from Forkhead transcription factor FOXO3 to report on the PI3K/Akt pathway activity. With perturbation to growth factor signaling, we observed a transient response in the cytosolic \(NADH:NAD^+\) redox state. In contrast, with partial inhibition of glycolysis by iodoacetate, individual cells varied substantially in their responses, and cytosolic \(NADH:NAD^+\) ratios oscillated between high and low states with a regular, approximately half-hour period, persisting for hours. These glycolytic NADH oscillations appeared to be cell-autonomous and coincided with the activation of the PI3K/Akt pathway but not the AMPK pathway. These results suggest a dynamic coupling between growth factor signaling and metabolic parameters. Overall, this thesis presents novel optical tools to assess metabolic dynamics – and to unravel the elaborate and complex integration of glucose metabolism and signaling pathways at the single cell level.
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Kosmacek, Elizabeth Anne Ianzini Fiorenza Mackey Michael A. "Live cell imaging technology development for cancer research." [Iowa City, Iowa] : University of Iowa, 2009. http://ir.uiowa.edu/etd/388.
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Chyan, Wen Ph D. Massachusetts Institute of Technology. "Fluorogenic probes for live-cell imaging of biomolecules." Thesis, Massachusetts Institute of Technology, 2018. http://hdl.handle.net/1721.1/118216.
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Thesis: Ph. D. in Biological Chemistry, Massachusetts Institute of Technology, Department of Chemistry, 2018.Cataloged from PDF version of thesis.
Includes bibliographical references (pages 231-249).
Fluorogenic probes, small-molecule sensors that unmask brilliant fluorescence upon exposure to specific stimuli, are essential tools for chemical biology. Probes that detect enzymatic activity can be used to illuminate the complex dynamics of biological processes at a level of spatiotemporal detail and sensitivity unmatched by other techniques. This dissertation describes the development of new fluorophore chemistries to expand our current fluorogenic probe toolkit and the subsequent application of these probes to study dynamic cell transport processes. Chapter 1. Enzyme-Activated Fluorogenic Probes for Live-Cell and In Vivo Imaging. Chapter 1 reviews recent advances in enzyme-activated fluorogenic probes for biological imaging, organized by enzyme classification. This review surveys recent masking strategies, different modes of enzymatic activation, and the breadth of current and future probe applications. Key challenges, such as probe selectivity and spectroscopic requirements, are described in this chapter along with therapeutic and diagnostic opportunities that can be accessed by surmounting these challenges. Chapter 2. Electronic and Steric Optimization of Fluorogenic Probes for Biomolecular Imaging. In many fluorogenic probes, the intrinsic fluorescence of a small-molecule fluorophore is masked by ester masking groups until entry into a cell, where endogenous esterases catalyze the hydrolysis of esters, generating fluorescence. The susceptibility of masking groups to spontaneous hydrolysis is a major limitation of these probes. Previous attempts to address this problem have incorporated auto-immolative linkers at the cost of atom economy and synthetic adversity. In this chapter, I report on a linker-free strategy that employs adventitious electronic and steric interactions in easy-to-synthesize probes. I find that halogen-carbonyl n-->[pi]* interactions and acyl group size are optimized in 2',7'-dichlorofluorescein diisobutyrate. This probe is relatively stable to spontaneous hydrolysis but is a highly reactive substrate for esterases both in vitro and in cellulo, yielding a bright, photostable fluorogenic probe with utility in biomolecular imaging. Chapter 3. Cellular Uptake of Large Monofunctionalized Dextrans. Dextrans are a versatile class of polysaccharides with applications that span medicine, cell biology, food science, and consumer goods. In Chapter 3, I apply the electronically stabilized probe described in Chapter 2 to study the cellular uptake of a new type of large monofunctionalized dextran that exhibits unusual properties: efficient cytosolic and nuclear uptake. This dextran permeates various human cell types without the use of transfection agents, electroporation, or membrane perturbation. Cellular uptake occurs primarily through active transport via receptor-mediated processes. These monofunctionalized dextrans could serve as intracellular delivery platforms for drugs or other cargos. Chapter 4. Paired Nitroreductase-Probe System to Quantify the Cytosolic Delivery of Biomolecules. Cytosolic delivery of large biomolecules is a significant barrier to therapeutic applications of CRISPR, RNAi, and biologics such as proteins with anticancer properties. In Chapter 4, I describe a new paired enzyme-probe system to quantify cytosolic delivery of biomolecules-a valuable resource for elucidating mechanistic details and improving delivery of therapeutics. I designed and optimized a nitroreductase fusion protein that embeds in the cytosolic face of outer mitochondrial membranes, providing several key improvements over unanchored reporter enzymes. In parallel, I prepared and assessed a panel of nitroreductase-activated probes for favorable spectroscopic and enzymatic activation properties. Together, the nitroreductase fusion protein and fluorogenic probes provide a rapid, generalizable tool that is well-poised to quantify cytosolic delivery of biomolecules. Chapter 5. Future Directions. This chapter outlines several future directions for expanding the scope of fluorogenic probes and developing new biological applications. Additionally, Chapter 5 is followed by an appendix describing a tunable rhodol fluorophore scaffold for improved spectroscopic properties and versatility. Overall, the work described in this thesis illustrates the power of enzyme-activated fluorogenic probes to provide fresh insight into dynamic biological processes, with direct implications for improved therapeutic delivery.
by Wen Chyan.
Ph. D. in Biological Chemistry
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Büchele, Benjamin. "Live Cell Imaging des Hepatitis C Virus Replikationskomplexes." [S.l. : s.n.], 2004. http://nbn-resolving.de/urn:nbn:de:bsz:25-opus-59102.
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Kosmacek, Elizabeth Anne. "Live cell imaging technology development for cancer research." Diss., University of Iowa, 2009. https://ir.uiowa.edu/etd/388.
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Live cell imaging is a unique tool for cellular research with a wide variety of applications. By streaming digital microscopic images an investigator can observe the dynamic morphology of a cell, track cell movement on a surface, and measure quantities or localization patterns of fluorescently labeled proteins or molecules. Digital image sequences contain a vast amount of information in the form of visually detectable morphological changes in the cell. We designed computer programs that allow the manual identification of visible events in live cell digital image sequences [Davis et al. 2007]. Once identified, the data are analyzed using algorithms to calculate the yield of individual events per cell over the time course of image acquisition. The sequence of event data is also constructed into directed acyclic graphs and through the use of a subgraph isomorphism algorithm we are able to detect specified patterns of events originating from a single cell. Two projects in the field of cancer research are here discussed that describe and validate the application of the event analysis programs. In the first project, mitotic catastrophe (MC) research [Ianzini and Mackey, 1997; Ianzini and Mackey, 1998; reviewed by Ianzini and Mackey, 2007] is enhanced with the addition of live cell imaging to traditional laboratory experiments. The event analysis program is used to describe the yield of normal or abnormal divisions, fusions, and cell death, and to detect patterns of reductive division and depolyploidization in cells undergoing radiation-induced MC. Additionally, the biochemical and molecular data used in conjunction with live cell imaging data are presented to illustrate the usefulness of combining biology and engineering techniques to elucidate pathways involved in cell survival under different detrimental cell conditions. The results show that the timing of depolyploidization in MC cells correlates with increased multipolar divisions, up-regulation of meiosis-specific genes, and the production of mononucleated cell progeny. It was confirmed that mononucleated cells are produced from multipolar divisions and these cells are capable of resuming normal divisions [Ianzini et al., 2009]. The implications for the induction of meiosis as a mechanism of survival after radiation treatment are discussed. In the second project, the effects of long-term fluorescence excitation light exposure are examined through measurements of cell division and cell death. In the field of live cell imaging, probably the most modern and most widely utilized technique is fluorescence detection for intracellular organelles, proteins, and molecules. While the technologies required to label and detect fluorescent molecules in a cell are well developed, they are not idealized for long term measurements as both the probes and excitation light are toxic to the cells [Wang and Nixon, 1978; Bradley and Sharkey, 1977]. From the event analysis data it was determined that fluorescence excitation light is toxic to multiple cell lines observed as the reduction of normal cell division, induction of cell death, and apparent morphological aberrations.
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Caporale, Chiara. "Luminescent Iridium Tetrazolato Markers for Live Cell Imaging." Thesis, Curtin University, 2018. http://hdl.handle.net/20.500.11937/70386.
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In this research, a series of iridium(III) tetrazolato complexes were synthesised and their photophysical and biological properties investigated. Both the cyclometalated and the ancillary ligands were systematically modified by substitution of functional groups or by increasing the extension of the conjugation. This approach allowed a more systematic rationalisation of the structure-activity relationship, highlighting how variations in the chemical structure and charge might influence the biological behaviour of these complexes.
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Danylchuk, Dmytro. "Environment-sensitive targeted fluorescent probes for live-cell imaging." Thesis, Strasbourg, 2021. http://www.theses.fr/2021STRAF012.
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Le ciblage, l'imagerie et le sondage spécifiques des membranes plasmiques et des organites intracellulaires peuvent être faits par des sondes fluorescentes à façon sensibles à la polarité. Ici, un nouveau fragment ciblant la membrane plasmique à été développé et testé dans cinq colorants cyanines, montrant d'excellentes performances en microscopie cellulaire et in vivo. Le fragment à été greffé à un fluorophore solvatochrome Prodan, donnant une sonde de membrane plasmique avec une sensibilité élevée à l'ordre lipidique. Le rouge de Nil, greffé aux fragments avec les chaînes alkyles C12 et C4, à donné deux sondes solvatochromes à membrane plasmique : NR12A pour la microscopie conventionnelle, et NR4A pour la microscopie à super-résolution PAINT. Le rouge de Nil avec des groupes ciblant les organites à donné un éventail de sondes sensibles à la polarité et à l'ordre lipidique dans les membranes des organites. Les sondes synthétisées trouveront des applications en bioimagerie, biologie cellulaire, biophysique ou mécanobiologieSpecific targeting, imaging and probing of cell plasma membranes and intracellular organelles can be addressed by rationally designed polarity-sensitive fluorescent probes. Here, a new efficient plasma membrane-targeting moiety was developed and tested in five cyanine dyes, showing excellent performance in cellular and in vivo microscopy. Next, the targeting moiety was grafted to a solvatochromic dye Prodan, yielding a plasma membrane probe with high lipid order sensitivity. Modifying a Nile Red using the moieties with varied alkyl chain lengths resulted in two solvatochromic plasma membrane probes: NR12A with high affinity to membranes for conventional microscopy, and NR4A, a low-affinity probe for PAINT super-resolution microscopy. Tethering Nile Red with organelle-targeted groups yielded an array of probes, able to sense polarity and lipid order in organelle membranes. The synthesized probes will find applications in bioimaging, cell biology, biophysics or mechanobiology
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Sörman, Paulsson Elsa. "Evaluation of In-Silico Labeling for Live Cell Imaging." Thesis, Umeå universitet, Institutionen för fysik, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-180590.
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Today new drugs are tested on cell cultures in wells to minimize time, cost, andanimal testing. The cells are studied using microscopy in different ways and fluorescentprobes are used to study finer details than the light microscopy can observe.This is an invasive method, so instead of molecular analysis, imaging can be used.In this project, phase-contrast microscopy images of cells together with fluorescentmicroscopy images were used. We use Machine Learning to predict the fluorescentimages from the light microscopy images using a strategy called In-Silico Labeling.A Convolutional Neural Network called U-Net was trained and showed good resultson two different datasets. Pixel-wise regression, pixel-wise classification, andimage classification with one cell in each image was tested. The image classificationwas the most difficult part due to difficulties assigning good quality labels tosingle cells. Pixel-wise regression showed the best result.
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Han, Hongqing. "Towards accurate and efficient live cell imaging data analysis." Doctoral thesis, Humboldt-Universität zu Berlin, 2021. http://dx.doi.org/10.18452/22324.
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Dynamische zelluläre Prozesse wie Zellzyklus, Signaltransduktion oder Transkription zu analysieren wird Live-cell-imaging mittels Zeitraffermikroskopie verwendet.
Um nun aber Zellabstammungsbäume aus einem Zeitraffervideo zu extrahieren, müssen die Zellen segmentiert und verfolgt werden können. Besonders hier, wo lebende Zellen über einen langen Zeitraum betrachtet werden, sind Fehler in der Analyse fatal: Selbst eine extrem niedrige Fehlerrate kann sich amplifizieren, wenn viele Zeitpunkte aufgenommen werden, und damit den gesamten Datensatz unbrauchbar machen.
In dieser Arbeit verwenden wir einen einfachen aber praktischen Ansatz, der die Vorzüge der manuellen und automatischen Ansätze kombiniert. Das von uns entwickelte Live-cell-Imaging Datenanalysetool ‘eDetect’ ergänzt die automatische Zellsegmentierung und -verfolgung durch Nachbearbeitung. Das Besondere an dieser Arbeit ist, dass sie mehrere interaktive Datenvisualisierungsmodule verwendet, um den Benutzer zu führen und zu unterstützen. Dies erlaubt den gesamten manuellen Eingriffsprozess zu rational und effizient zu gestalten. Insbesondere werden zwei Streudiagramme und eine Heatmap verwendet, um die Merkmale einzelner Zellen interaktiv zu visualisieren. Die Streudiagramme positionieren ähnliche Objekte in unmittelbarer Nähe. So kann eine große Gruppe ähnlicher Fehler mit wenigen Mausklicks erkannt und korrigiert werden, und damit die manuellen Eingriffe auf ein Minimum reduziert werden. Die Heatmap ist darauf ausgerichtet, alle übersehenen Fehler aufzudecken und den Benutzern dabei zu helfen, bei der Zellabstammungsrekonstruktion schrittweise die perfekte Genauigkeit zu erreichen. Die quantitative Auswertung zeigt, dass eDetect die Genauigkeit der Nachverfolgung innerhalb eines akzeptablen Zeitfensters erheblich verbessern kann. Beurteilt nach biologisch relevanten Metriken, übertrifft die Leistung von eDetect die derer Tools, die den Wettbewerb ‘Cell Tracking Challenge’ gewonnen haben.Live cell imaging based on time-lapse microscopy has been used to study dynamic cellular behaviors, such as cell cycle, cell signaling and transcription. Extracting cell lineage trees out of a time-lapse video requires cell segmentation and cell tracking. For long term live cell imaging, data analysis errors are particularly fatal. Even an extremely low error rate could potentially be amplified by the large number of sampled time points and render the entire video useless. In this work, we adopt a straightforward but practical design that combines the merits of manual and automatic approaches. We present a live cell imaging data analysis tool `eDetect', which uses post-editing to complement automatic segmentation and tracking. What makes this work special is that eDetect employs multiple interactive data visualization modules to guide and assist users, making the error detection and correction procedure rational and efficient. Specifically, two scatter plots and a heat map are used to interactively visualize single cells' visual features. The scatter plots position similar results in close vicinity, making it easy to spot and correct a large group of similar errors with a few mouse clicks, minimizing repetitive human interventions. The heat map is aimed at exposing all overlooked errors and helping users progressively approach perfect accuracy in cell lineage reconstruction. Quantitative evaluation proves that eDetect is able to largely improve accuracy within an acceptable time frame, and its performance surpasses the winners of most tasks in the `Cell Tracking Challenge', as measured by biologically relevant metrics.
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Flaccavento, Giselle. "Imaging tools for live cell micro-irradiation survival studies." Thesis, University of Oxford, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.589627.
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Micro-irradiation systems are used to analyse the effect of ionizing radiation at the cellular and tissue level, targeting individual cells within a population with a controlled low dose. Cell survival experiments using micro-irradiation systems are limited by factors including: 1) the radiation attenuation and optical properties of the chosen cell dish substrate, 2) the registration of the cell dish before and after irradiation or between multiple imaging modalities and 3) the analysis of the cell or colony growth after irradiation. In this thesis, a set of tools have been developed to improve micro-irradiation experiments and to increase the accuracy of information provided by the cell survival data. The first contribution, the substrate cell dish evaluation, provides a set of characteristics defining the substrates used for micro-irradiation experiments based on minimal energy loss and optical clarity using unstained cell imaging. The second contribution was the development of a novel and low cost fiducial marking device for micro-irradiation experiments using an 808 nm laser and providing marks suitable for imaging with multiple modalities. The minimum focused spot diameter was calculated as 22.9 urn and the device was used to create fiducial marks with diameters ranging from 20 urn to 130 urn. The third contribution, the development of a cell counting methodology for use with a lens-free imaging device, has been shown to accurately count thousands of cells suitable for immediate analysis. Approximately 1000 cell colonies, containing 17 729 cells on 11 cell dishes were used for testing and training for automatic cell counting. Validation of the cell counting method showed that 76% and 89% of the cell colonies were counted within a ± 20% and ± 30% error of the ground truth, respectively. Further development of the fiducial marking device, by modifying the choice of laser and making it suitable for multiple types of cell dish substrates, would increase the applications of the device. Development of the cell counting methodology for different cells line, and for cells grown on multiple types of substrates, would make the system suitable for analysis of a wide variety of cell survival studies. The cell counting methodology, applied to the CyMap lens-free imaging device, has the potential to be an extremely useful and cost effective tool for cell survival studies.
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Saurabh, Saumya. "Ultra-Photostable Genetically Targeted Fluoromodules for Live Cell Imaging." Research Showcase @ CMU, 2014. http://repository.cmu.edu/dissertations/1020.
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Khorshidi, Mohammad Ali. "Live Single Cell Imaging and Analysis Using Microfluidic Devices." Doctoral thesis, KTH, Proteomik och nanobioteknologi, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-129278.
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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.QC 20130927
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Miura, Haruko. "Live-Cell Imaging of Stress Signaling Dynamics in a Cell Fate Decision." Kyoto University, 2019. http://hdl.handle.net/2433/236635.
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Babic, Julien. "New microfluidic systems for controlling the cell microenvironment during live-cell imaging." Thesis, Rennes 1, 2017. http://www.theses.fr/2017REN1B047/document.
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Connaître en temps réel la réponse et le comportement des cellules et organismes modèles suite à des changements de leur environnement, ou à des modulations de leurs fonctions biologiques est devenu essentiel dans les sciences du vivant. Ces réponses nous permettent ensuite de comprendre les mécanismes qui régissent le fonctionnement des cellules vivantes, avec des implications en recherche fondamentale, appliquée et biomédicale. Un des plus gros défis technologiques reste le contrôle des paramètres environnementaux en microscopie haute résolution. De nos jours, aucun système ne permet de réguler un ensemble complexe de paramètres de manière précise, dynamique et simultanée tout en observant les cellules dans leur environnement. L’objectif de ma thèse est de mettre au point un tel dispositif permettant a minima une régulation fine de la température, de la composition du milieu, et notamment de la concentration de divers drogues. Ce système doit être compatible avec les applications les plus poussées en microscopie photonique. Mon approche au cours de ma thèse pour élaborer un tel système est l’utilisation de la microfluidique. En effet, c’est la seule technologie qui puisse de réaliser un tel multiplexage. Elle permet de manipuler des petites quantités de fluide à travers un système contenant des canaux de dimensions allant du micromètre au centimètre. Cet ordre de grandeur des canaux constitue un atout majeur (réduction de la consommation des réactifs, réduction des couts, cinétiques des réactions chimiques et biologiques élevées, temps de diffusion court, etc.) et permet d’allier les expériences biologiques à la microscopie. Mon objectif est de concevoir une puce microfluidique qui représentera un pas technologique majeur et ouvrira de nouvelles possibilités de rechercheMonitoring in real-time the response of cells and model organisms to the changes in their environment or to modulations of their biological functions has become essential in life sciences. One of the main technical challenges for biologists is the precise and dynamic control of various environmental parameters while doing high-resolution microscopy. My thesis consists of building a robust and versatile system, dedicated to live-cell imaging that will be compatible with adherent and non adherent models, that could provide a precise and simultaneous control of 1) the temperature, 2) the media exchanges and 3) the drug concentration while doing photonic microscopy. My approach is to use microfluidics, which is the best candidate in order to achieve this system and provides all the necessary controls of micro-scaled volumes for culturing, maintaining or analyzing cells. It produces miniaturized systems used as tools for biological experiments, in which channels of a micro-scaled dimension are used for the fluid circulation. The laminar flow in these chips allows fast molecule diffusion as well as fast temperature diffusion. Because of the high surface to volume ratio, the consumption of reagents is reduced, and media switches can be fast. This system will represent a major technical and beneficial step and will open new possibilities of research in biology
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Sauer, Anna Magdalena. "Live-cell imaging of drug delivery by mesoporous silica nanoparticles." Diss., lmu, 2011. http://nbn-resolving.de/urn:nbn:de:bvb:19-138222.
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Rosa, Stefanie. "Chromatin dynamics in Arbidopsis development: a live cell imaging approach." Doctoral thesis, Universidade Nova de Lisboa. Instituto de Tecnologia Química e Biológica, 2011. http://hdl.handle.net/10362/6847.
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Dissertation presented to obtain the Ph.D degree in BiologyThe proper development of multicellular organisms demands the distinct specification of a variety of specialized cell types. While this is one of the oldest statements of developmental genetics, how different patterns of gene expression are established in genetically identical cells and maintained during somatic cell divisions is still an active topic of research. Chromatin structure is now recognized to regulate gene activity playing a crucial role in cell differentiation and development. Chromatin is not simply a packaging tool but a dynamic entity that reflects the regulatory cues necessary to program appropriate cellular pathways. There are several ways by which chromatin structure can be remodelled. These mechanisms include DNA-methylation, post-translational modifications of histone proteins, histone variants and, nuclear localization. While the dynamic nature of chromatin structure has been previously described its biological function and repercussions on development are only now beginning to be revealed. In this work we used in vivo microscopy techniques to assess how different aspects of chromatin organization play a role on various aspects of development.(...)
A bolsa de doutoramento com a referência SFRH/BD/23202/2005 foi atribuída pela Fundação para a Ciência e Tecnologia (FCT), no âmbito do Quadro Comunitário de Apoio, comparticipado pelo Fundo Social Europeu (FSE).
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Forsgren, Edvin. "Deep Learning to Enhance Fluorescent Signals in Live Cell Imaging." Thesis, Umeå universitet, Institutionen för fysik, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-175328.
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Yao, Zhizhong. "Using Live Cell Imaging to Probe Biogenesis of the Gram-Negative Cell Envelope." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10230.
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In Gram-negative bacteria, the three-layered cell envelope, including the cell wall, outer and inner membranes, is essential for cell survival in the changing, and often hostile environments. Conserved in all prokaryotes, the cell wall is incredibly thin, yet it functions to prevent osmotic lysis in diluted conditions. Based on observations obtained by genetic and chemical perturbations, time-lapse live cell imaging, quantitative imaging and statistical analysis, Part I of this dissertation explores the molecular and physical events leading to cell lysis induced by division-specific beta-lactams. We found that such lysis requires the complete assembly of all essential components of the cell division apparatus and the subsequent recruitment of hydrolytic amidases. We propose that division-specific beta-lactams lyze cells by inhibiting FtsI (PBP3) without perturbing the normal assembly of the cell division machinery and the consequent activation of cell wall hydrolases. On the other hand, we demonstrated that cell lysis by beta-lactams proceeds through four physical phases: elongation, bulge formation, bulge stagnation and lysis. Bulge formation dynamics is determined by the specific perturbation of the cell wall and outer membrane plays an independent role in stabilizing the bulge once it is formed. The stabilized bulge delays lysis, and allows escape and recovery upon drug removal. Asymmetrical in structure and unique to Gram-negative bacteria, outer membrane prevents the passage of many hydrophobic, toxic compounds. Together with inner membrane and the cell wall, three layers of the Gram-negative cell envelope must be well coordinated throughout the cell cycle to allow elongation and division. Part II of this dissertation explores the essentiality of the LPS layer, the outer leaflet of the outer membrane. Using a conditional mutant severely defective in LPS transport, we found that mutations in the initiation phase of fatty acid synthesis suppress cells defective in LPS transport. The suppressor cells are remarkably small with a 70% reduction in cell volume and a 50 % reduction in growth rate. They are also blind to nutrient excess with respect to cell size control. We propose a model where fatty acid synthesis regulates cell size in response to nutrient availability, thereby influencing growth rate.Chemistry and Chemical Biology
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Smith, David. "Process monitoring and control using live cell imaging for the manufacturing of cell therapies." Thesis, Loughborough University, 2014. https://dspace.lboro.ac.uk/2134/16063.
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Regenerative medicine (RM) represents a promising enabling technology to revolutionize healthcare. This said there are still major gaps between the commercial promise and the reality of the cell therapy sector of regenerative medicine. There is consensus to develop high through-put, automated technologies for the manufacture of RM products. Imaging methods will have the capacity to contribute to this technological gap for cell therapies and are particularly attractive to provide non-destructive monitoring with high spatial and temporal resolution. This work applied an automated, non-invasive phase contrast imaging platform (Cell-IQ) to measure, analyse and ultimately quantify image derived metrics for human embryonic stem cells (hESCs) and haematopoietic stem cells (HSCs) as part of the colony forming unit (CFU) assay. This work has shown through thresholding and machine vision identification technology, imaging has the ability to improve the precision of current evaluation methods for cell culture, providing novel information regarding culture state and show image derived metrics to be predictive of future culture state. Building on this, differentiation through the addition of a growth factor cocktail highlighted how in-process monitoring enables protocol optimisation. After equilibrating the Cell-IQ incubator to a standard incubator, the progress of the CFU assay was monitored and image metrics representative of colony phenotype were analysed. Cell count, distance between cells and cell migration within individual colonies were identified to be informative and provide a degree of colony phenotype separation. Quantitative, novel, image derived metrics were identified that improve reliability through computer automation, cost by removing user verification and time by reducing the assay time from 14 days to 7 days. Non-invasive imaging provides a fantastic opportunity to create bespoke sampling frequencies to achieve desired precision for manufacturing cell therapies, this work has developed and shown improvement and a level of control to current culture process for ESCs and HSCs.
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Auciello, Giulio. "Analysis of FGF receptor signalling and trafficking by live-cell imaging." Thesis, University of Birmingham, 2013. http://etheses.bham.ac.uk//id/eprint/4650/.
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Fibroblast growth factor receptors (FGFRs) regulate fundamental cellular processes, including proliferation, differentiation and angiogenesis and have emerged as growth factor receptors central to oncogenesis. This study developed a live-cell assay system for studying FGFR endocytosis and trafficking by employing both confocal and total internal reflection fluorescence (TIRF) microscopy in cells expressing a previously characterised GFP-tagged FGFR2 construct. Data from this work have demonstrated that endocytosis of activated FGFR occurs through clathrin-mediated endocytosis. Interestingly, FGF treatment also significantly increased the number of CCPs as well as the number of clathrin-mediated endocytic events. However, treatment of cells with the Src family inhibitor Dasatinib or depletion of Src kinase target Eps8, prevents the FGF induced increase in plasma membrane clathrin and reduces the internalization of FGFR. This study also shows that both Src and Eps8 are required for receptor to exit from EEA I positive peripheral compartment into the Rab 1 1 positive PNRC. Eps8 depletion also inhibits the early phases of ERK activation in response to FGFR activation, placing this signalling event early in the trafficking pathway of the receptor. Thus, these results have identified the endocytic pathway for endocytosis of FGFR2 and described Eps8 and Src as key mediators of the early phases of activated FGFR trafficking and signalling.
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Han, Hongqing [Verfasser]. "Towards accurate and efficient live cell imaging data analysis / Hongqing Han." Berlin : Humboldt-Universität zu Berlin, 2021. http://d-nb.info/1226153445/34.
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Hailey, Dale W. "Live cell imaging to study the assembly and fate of autophagosomes." College Park, Md.: University of Maryland, 2008. http://hdl.handle.net/1903/8796.
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Thesis (Ph. D.) -- University of Maryland, College Park, 2008.Thesis research directed by: Dept. of Cell Biology and Molecular Genetics. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
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Dodgson, Lauren. "Dissecting the molecular mechanisms of Drosophila border cell migration using time-lapse live cell imaging." Thesis, University of Liverpool, 2013. http://livrepository.liverpool.ac.uk/16293/.
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Dissection of the cellular dynamics and molecular pathways that drive collective cell migration is necessary to better understand cellular rearrangements that underpin normal development, as well as disease states such as cancer metastasis. Border cell migration in the Drosophila ovary has proven to be a good model of invasive cell migration, because of its genetic tractability, and also because recent advances in culturing egg chambers ex vivo have facilitated live cell imaging in this system. The aim of this thesis was to further develop and implement live cell imaging approaches, and to apply these to characterise the role of Pico, the Drosophila Mig10/RIAM/Lpd (MRL) protein in border cell migration. MRL proteins are known to regulate actin dynamics, but their role in epithelial cell migration had not been established. Through careful optimisation, suitable approaches were developed for: medium preparation; dissection and mounting of egg chambers; acquisition of images by confocal microscopy. A fluorescently-labelled reporter strain with improved optical properties was generated to monitor actin dynamics, and a number of other reporters were characterised, either alone or in combination, to determine their behaviour and effect on migration. After trialling several analytical tools and quantitative methods, a streamlined approach to analysing the image data was developed allowing: tracking of border cell migration in four dimensions (XYZ and time) to obtain information about behaviour of the migratory cells; measurement of cellular protrusion dynamics to obtain mechanistic insight into why cellular dynamics might change in different genetic backgrounds. Finally, these approaches were applied to the characterisation of Pico and its interacting partner SCAR, demonstrating that pico affects border cell migration through the modulation of actin protrusion dynamics in a SCAR-dependent manner.
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Mickler, Frauke Martina. "Live-cell imaging elucidates cellular interactions of gene nanocarriers for cancer therapy." Diss., Ludwig-Maximilians-Universität München, 2013. http://nbn-resolving.de/urn:nbn:de:bvb:19-165829.
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Linnik, Volha. "Functional analysis of a plant virus replication 'factory' using live cell imaging." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/4639.
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Plant viruses have developed a number of strategies that enable them to become obligate intracellular parasites of many agricultural crops. Potato virus X (PVX) belongs to a group of positive-sense, single-stranded plant RNA viruses that replicate on host membranes and form elaborate structures known as viral replication complexes (VRCs) that contain viral RNA (vRNA), proteins and host cellular components. VRCs are the principal sites of viral genome replication, virion assembly and packaging of vRNA for export into neighbouring cells. For many animal viruses, host membrane association is crucial for RNA export. For plant viruses, it is not yet known how vRNA is transported to and through plant plasmodesmata. PVX encodes genetic information required for its movement between cells; three viral triple gene block (TGB) movement proteins and a viral coat protein are essential for viral trafficking. This research project studies the relationship between PVX and its host plants, Nicotiana benthamina and Nicotiana tabacum. A particular focus of this project is exploration of the structural and functional significance of the PVX VRC and how the virus recruits cell host components for its replication and movement between cells. The role of specific viral proteins in establishing the VRC, and the ways in which these interact with host organelles, was investigated. A combination of different approaches was used, including RNA-binding dyes and a Pumilio-based bimolecular fluorescence complementation assay for detection of the vRNA, fluorescent reporters for virusencoded proteins, fluorescent reporters for host organelles involved in viral replication, and also transgenic tobacco plants expressing reporters for specific plant components (endoplasmic reticulum, Golgi, actin, microtubules and plasmodesmata). In addition, mutagenesis was used to study the functions of individual viral proteins in replication and movement. All of these approaches were combined to achieve live-cell imaging of the PVX infection process. The PVX VRC was shown to be a highly compartmentalised structure; (+)-stranded vRNA was concentrated around the viral TGB1 protein, which was localised in discrete circular compartments within the VRC while coat protein was localised to the external edges of the VRC. The vRNA was closely associated with host components (endoplasmic reticulum and actin) shown to be involved in the formation of the VRC. The TGB2/TGB3 viral proteins were shown to colocalise with the host endomembranes (ER) and to exit these compartments in the form of motile granules. vRNA, TGB1, TGB2 and CP localised to plasmodesmata of the infected cells. TGB1 was shown to move cell-to-cell and recruit ER, Golgi and actin in the absence of viral infection. In the presence of virus, TGB1 targeted the VRCs in several neighbouring cells. A model of PVX replication and movement is proposed in which TGB1 functions as a key component for recruitment of host components into the VRC to enable viral replication and spread.
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Zhai, Weichao. "Microfluidics and live imaging advances : applications in host/pathogen, immunity and stem cell single cell phenotyping." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/277189.
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Live single-cell imaging has emerged as an advanced single-cell study tool for approaching a quantitative understanding of many biological questions in recent years. In previous cell studies using bulk cell measurements, the population averages can miss the information from cell to cell variability and mask the underlying signaling networks and mechanisms. Currently, some single cell analysis methods, including but not limited to, live single-cell imaging experiments that built around a fluorescent imaging setup and microfluidic devices enable the measurement and analysis of cell dynamics and responses of single cells across a population and across time. Furthermore, by changing the cells’ environmental conditions in well controlled ways, e.g. balanced steady growth, or temporal pulses, live single-cell imaging can record the cellular behaviors corresponding to these changes in exquisite details. An important question of current interest in both developmental, stem cell and cancer biology is the question of epigenetic differentiation. Continuous long-term live single-cell observations offer insights into the molecular control of cell fate. However, maintaining the imaged cells in a healthy state remains a major challenge. One of our aims in this work was to develop a semi-automated single-cell live imaging and analysis platform to obtain dynamic information of the cellular processes. An imaging incubator that controls and regulates the environmental conditions of the imaged cells also had to be designed and tested. In this thesis, I address the key design considerations of developing a single-cell live imaging platform and demonstrate the capability of this technology through three case studies. To test the design and fabrication of microfluidic devices and micro-valves in imaging malaria infected red blood cells (iRBCs), I recorded the flow of iRBCs through microfluidic channels and constrictions in Chapter 3. Our results illustrate the behaviors of iRBCs with different flow rates and the potential to offer dynamic control in studying the infection probability of iRBCs by implementing the micro-valve system. In order to develop a more adaptable live cell imaging platform, we further developed our semi-automated imaging software and in house built imaging incubator to explore the link between proliferation and differentiation of CD4+ T cells in Chapter 4. By using cells expressing an IL-13-GFP reporter, we distinguished between differentiating and non-differentiating CD4+ T cell population and demonstrated a positive association between cycling differentiation of CD4+ T cells. In Chapter 5, we incorporated the FUCCI cell reporter system in our single cell live imaging system to reveal the effect of different media conditions on the cell cycle progression and cell fate choices of mouse embryonic stem (mES) cells. By improving different factors such as longer pre-incubation time before imaging and exchanging media during the experiments, we maintained a healthy state of mES cells during live cell imaging for extended periods. We observed significant differences in time between divisions of mES cells cultured in 2i +LIF and serum + LIF media, and also small but significant differences in durations of sub-cell cycle phases (G1,G1/S,S/G2/M) between the two media conditions. We further applied this imaging setup to study the behaviors of differentiating mES cells in vitro, and observed lengthening of the G1 phase for both 2i-LIF and serum-LIF cells in agreement with literature. Overall, our semi-automated single cell imaging platform not only offers adjustable intervals between fluorescent imaging, but also provides a constant temperature and gas feeding devices that allows the cells to proliferate for extended microscope imaging. Commercially produced incubators that fit onto the microscope stage and satisfied all requirements in restriction of the cell movement, gas feeding, temperature regulation and optical accessibility are not easily available. Thus, there exists a significant potential for our imaging setup to provide a versatile and adaptable live cell imaging platform for both academia and industrial researchers.
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Moreira, Severina. "Live imaging and genetic studies of inflammatory cell migration in Drosophila melanogaster embryos." Thesis, University of Bristol, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.508069.
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Mizusawa, Keigo. "Development of Fluorescent Turn-on Self-assembled Nanoprobes for Imaging Specific Proteins under Live Cell Conditions." 京都大学 (Kyoto University), 2013. http://hdl.handle.net/2433/174966.
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Yatsuzuka, Kenji. "Live-cell imaging of multiple endogenous mRNAs permits the direct observation of RNA granule dynamics." Kyoto University, 2019. http://hdl.handle.net/2433/242400.
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Knopp, Marcus. "Analysis of spine plasticity in CA1 hippocampal pyramidal neurons employing live cell nanoscopic imaging." Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-173975.
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In der Großhirnrinde von Säugetieren befindet sich die Mehrheit erregender Synapsen auf Dornfortsätzen, kleinen dendritischen Ausbuchtungen, die in Größe und Form stark variieren. Die Auslösung aktivitätsabhängiger synaptischer Langzeitplastizität geht mit strukturellen Veränderungen dendritischer Dornen einher. Da das beugungsbegrenzte Auflösungsvermögen konventioneller Lichtmikroskope nicht ausreicht um die Morphologie der Dornen verlässlich zu untersuchen, stellte die Elektronenmikroskopie bisher das wichtigste bildgebende Verfahren zur Erforschung von struktureller Plastizität dar, blieb dabei jedoch auf die Betrachtung fixierter Gewebeproben beschränkt. Die Anwendung hochauflösender Laser-Raster-Mikroskopie mit Stimulierter-Emissions-Auslöschung hat es mir möglich gemacht, die Dynamik dendritischer Dornenmorphologie in lebenden Zellen zu studieren. Die N-Methyl-D-Aspartat-Rezeptor-abhängige Langzeitpotenzierung von Pyramidenzellen der Cornu-Ammonis Region 1 des Hippocampus bildete dabei den Mechanismus, welcher plastische Veränderungen hervorrief. Nach Potenzierung exzitatorischer Synapsen durch die lokale Ultraviolett-Photolyse von caged-Glutamat wurde ein starker, vorübergehender Anstieg des Anteils dendritischer Dornen mit sichelförmigen Köpfen und ein leichter, anhaltender Zuwachs an pilzförmigen Dornfortsätzen über einen Zeitraum von 50 Minuten beobachtet. Meine Untersuchungen ergänzen frühere Studien zur Wechselbeziehung zwischen synaptischer Potenzierung und struktureller Plastizität dendritischer Dornen und korrespondieren mit dem aktuellen Kenntnisstand der zu Grunde liegenden molekularen Mechanismen.The majority of excitatory synapses in the cortex of mammalian brains is situated on dendritic spines, small protrusions, heterogeneous in size and shape. The induction of activity-dependent long-term synaptic plasticity has been associated with changes in the ultrastructure of spines, particularly in size, head shape and neck width. Since the dimensions of dendritic spines are at the border of the diffraction-limited resolving power of conventional light microscopes, until recently, electron microscopy on fixed tissue constituted the primary method for investigations on spine morphology. I have employed live cell stimulated emission depletion imaging to analyse spine motility and structural transitions in response to n-methyl-d-aspartate receptor dependent long-term potentiation over time at super-resolution in Cornu Ammonis area 1 pyramidal neurons of the hippocampus. Local induction of long-term potentiation via ultraviolet photolysis of caged glutamate facilitated a strong transient increase in the proportion of spines with curved heads and a subtle persistent growth in the amount of mushroom spines over a time course of 50 minutes. My findings reinforce previous investigations on the relation of synaptic potentiation and spine motility, and are in good agreement with the current knowledge of the molecular mechanisms underlying long-term plasticity.
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Zhang, Yun. "Real time imaging of live cell ATP leaking or release events by chemiluminescence microscopy." [Ames, Iowa : Iowa State University], 2008.
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Bhat, Anayat [Verfasser]. "Live Cell Fluorescence Imaging of Nucleotide Dynamics : ATP Hydrolysis and DNA Damage Response / Anayat Bhat." Konstanz : KOPS Universität Konstanz, 2021. http://d-nb.info/1229351094/34.
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Altenbach, Kirsten. "Development and analysis of recombinant fluorescent probes for use in live cell imaging of filamentous fungi." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/4739.
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The molecular cloning and subsequent engineering of the green fluorescent protein (GFP) of the jellyfish Aequoria victoria allowed a novel approach to the investigation of cell signalling. GFP and its mutants can now not only be used to target specific organelles in living cells but also function as a basis for a variety of sensors for biologically important ions and molecular interactions. GFP-based Ca2+- sensors have been successfully used for studies in mammalian and plant cells. In filamentous fungi, however, they have not yet been reported to work. Since only little is known about calcium signalling in filamentous fungi, this project aimed to improve existing GFP-based Ca2+- sensors by exchanging the original fluorophores for improved versions and expressing those in the filamentous fungus Aspergillus niger. During this project, the donor and acceptor fluorophores of 3 existing Ca2+-FRETprobes based on cameleons and troponin C-sensors, have been changed, 2 novel positive FRET controls have been designed and these , as well as donor and acceptor fluorophores alone, have been expressed in the filamentous fungus Aspergillus niger. The probes were assessed using different imaging techniques, such as conventional confocal laser scanning microscopy (CLSM), fluorescence lifetime imaging microscopy (FLIM) and spectral imaging using a Leica TSC SP5 confocal and IRIS, a novel spectral imaging device designed at Heriot Watt University. Problems were encountered that prevented FRET analysis using CLSM and IRIS. These were due mainly to the difference in expression level of the constructs and the distribution of the emission bandpasses of the IRIS system. Analysis of the spectral data obtained on the Leica confocal system and analysis of the FLIM results, however, revealed significant differences between the donor only and the positive FRET controls. Spectra of the positive FRET controls and the Ca2+-sensitive probes showed emission peaks of both the donor and the acceptor fluorophores upon excitation of the donor fluorophore alone while analysis of the FLIM results revealed an additional decay component in the positive FRET controls. Both results are very strong indicators that we can detect FRET in living hyphae of Aspergillus niger transformed with the probes designed during this project.
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Prusicki, Maria Ada [Verfasser], and Arp [Akademischer Betreuer] Schnittger. "Live cell imaging of meiosis in anthers of Arabidopsis thaliana / Maria Ada Prusicki ; Betreuer: Arp Schnittger." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2019. http://d-nb.info/1192442814/34.
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Maruno, Takahisa. "Visualization of stem cell activity in pancreatic cancer expansion by direct lineage tracing with live imaging." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/265166.
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京都大学新制・論文博士
博士(医学)
乙第13427号
論医博第2231号
新制||医||1053(附属図書館)
京都大学大学院医学研究科医学専攻
(主査)教授 松田 道行, 教授 渡邊 直樹, 教授 川口 義弥
学位規則第4条第2項該当
Doctor of Medical Science
Kyoto University
DFAM
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Prusicki, Maria Ada Verfasser], and Arp [Akademischer Betreuer] [Schnittger. "Live cell imaging of meiosis in anthers of Arabidopsis thaliana / Maria Ada Prusicki ; Betreuer: Arp Schnittger." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2019. http://nbn-resolving.de/urn:nbn:de:gbv:18-98605.
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Monypenny, James Edward. "Development of quantitative live cell imaging techniques and their applications in the study of inter-cellular communication and Sarcoma cell motility." Thesis, University College London (University of London), 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.406165.
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Bornheimer, Scott Joseph. "Spatial and temporal regulation of G-protein signaling elucidated by computational modeling and live cell FRET imaging." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2008. http://wwwlib.umi.com/cr/ucsd/fullcit?p3308008.
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Thesis (Ph. D.)--University of California, San Diego, 2008.Title from first page of PDF file (viewed June 12, 2008). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.
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White, Katharine Alice. "Rational design and directed evolution of probe ligases for site-specific protein labeling and live-cell imaging." Thesis, Massachusetts Institute of Technology, 2012. http://hdl.handle.net/1721.1/78438.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2012.Cataloged from PDF version of thesis.
Includes bibliographical references.
Chemical fluorophores have superior photophysical properties to fluorescent proteins and are much smaller. However, in order to use these probes for live-cell protein imaging, highly specific labeling methods are required. Here, we will describe three efforts to re-engineer the E. coli enzyme, lipoic acid ligase (LplA), to catalyze the ligation of small-molecule probes onto recombinant proteins. We call this collection of methods the PRIME (PRobe Incorporation Mediated by Enzymes) methodologies. First, we describe the structure-guided mutagenesis of LplA and the identification of an LplA variant that can ligate a blue coumarin fluorophore onto a 13-amino acid LplA acceptor peptide (LAP2). This "coumarin ligase" can be used to image cellular proteins with high specificity, sensitivity, and minimal perturbation of the biology of the protein of interest. We also demonstrate how subpopulations of a protein of interest can be labeled using genetically targeted coumarin ligase. Second, we describe our attempts to use yeast display evolution and fluorescence activated cell sorting (FACS) to evolve a truncated LplA enzyme. The original truncated enzyme had severely decreased activity for LplA's natural substrate, lipoic acid. We created a 107 library of LplA mutants and, after four rounds of selection, produced a truncated LplA mutant with lipoylation activity equivalent to full-length LplA. We next sought to evolve activity for an unnatural small molecule probe, but found that this strategy was limited by both increased hydrophobic probe sticking when using the truncated enzyme and some enzyme-dependent nonspecificity. Finally, from a library of 107 LplA mutants, we evolved a full-length LplA capable of ligating an unnatural picolyl azide (pAz) substrate. We demonstrated improved activity of the "pAz ligase" in the secretory pathway and cell surface, two regions where coumarin ligase is inactive. This enzyme can also be used to image cell surface protein-protein interactions as well as label proteins as they are trafficked through the endoplasmic reticulum. These probe ligases will be useful tools for cell biologists interested in studying protein function or protein-protein interactions in the context of living cells.
by Katharine Alice White.
Ph.D.
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Gustafsson, Linnéa. "Internalisation of antigen-adjuvant conjugate in human dendritic cells : An assay development for using live cell imaging." Thesis, Uppsala universitet, Institutionen för farmaceutisk biovetenskap, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-434224.
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Introduction: Cancer vaccines are a therapeutic approach to initiate an antigen specific cytotoxic immune responses against tumors. Cancer vaccines are composed by an antigen (tumor peptide) and adjuvant. A peptide in combination with adjuvants effectively activate dendritic cells (DCs), the most efficient antigen presenting cells in our immune system. DCs prime and activate CD8+ cytotoxic T cells which generates an antigen specific response.Aim: Developing an assay to study the internalisation rout of an antigen-adjuvant conjugate in human dendritic cells by using live cell imaging. Method: Immobilisation of cells is necessary for the ability to perform live cell imaging for several hours. The immobilisation ability of three coatings, collagen type I, fibronectin and matrigel, at different concentrations were evaluated by using live cell imaging in a fluorescence microscope. The potential induction of activation of the cells were evaluated by using flow cytometry and ELISA. Results: Immature DCs internalise antigen-adjuvant conjugate more efficiently than mature and activated DCs. Therefore, it is important that the coating do not induce activation. Cells must also be immobilised for the possibility of long term detection. Collagen type I immobilised cells and induced activation in all investigated concentrations. Fibronectin and matrigel had concentration-dependent abilities to immobilise the cells. Matrigel did not activate the cells whilst fibronectin was concentration dependent. Conclusion: Matrigel immobilise the cells which enables long term single cell imaging without activation.
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Panday, Namuna. "Scanning Ion Conductance Microscopy for Single Cell Imaging and Analysis." FIU Digital Commons, 2017. http://digitalcommons.fiu.edu/etd/3477.
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Most biological experiments are performed on an ensemble of cells under the assumption that all cells are identical. However, recent evidence from single cells studies reveals that this assumption is incorrect. Individual cells within the same generation may differ dramatically, and these differences have important consequences for the health and function of the entire living body. I have used Scanning Ion Conductance Microscopy (SICM) for imaging and analysis of topographical change of single cell membrane, which is difficult to be revealed by optical microscopes. Morphological change in the fixed and live HeLa cell membrane during endocytosis of conjugated polymer nanoparticles was studied. Results demonstrated SICM is a powerful tool to study the interaction between nanoparticle and cell membrane during internalization of nanoparticles through the membrane. This research can improve our fundamental understanding of cellular behavior and will be helpful for drug delivery applications.
Based on conventional SICM, we have developed a novel method to simultaneous map the topography and potential distributions of the single living cells membranes. At the first step, multifunctional nanopipettes (nanopore/nanoelectrode) have been fabricated and characterized. To demonstrate the potential sensing capability and understand the mechanism, I measured the ionic current and local electric potential change during translocation of 40 nm charged gold nanoparticles. Our results reveal the capability of the multifunctional probe for the highly sensitive detection of the ionic current and local electrical potential changes during the translocation of the charged entity through the nanopore. From the potential change, we revealed the dynamic assembly of GNPs before entering the nanopore. The experimental results are also nicely explained by the finite element method based numerical simulation results.
At the second step, I have measured the surface potential of living cell membrane at selected locations. Very recently, I have obtained results to show that we can map the extracellular membrane potential distribution of the complicated living cell membrane with sub-micron spatial resolution.This new imaging technique can help biologist to explore the extracellular potential distribution of varieties of cells quantitatively.These studies will have impacts on several biomedical applications such as regenerative repair and cancer treatment.
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Ali, Rizwan. "Live Cell Imaging of Intracellular Uptake of Contaminant Molecules (B[a]P) and its Effects on Different Cellular Compartments." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2012. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-91967.
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Exposure of hepatoma cell lines to the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P) is serving as a model for a systems biological study concerning the response of cells to contaminant molecules. Several aspects of the cellular distribution of the aryl hydrocarbon receptor (AhR) and its ligand B[a]P have been addressed by different live cell imaging techniques: The intracellular distribution of the B[a]P/AhR complex is visualized by means of confocal laser scaning microscopy (cLSM) and the intracellular transport rates of the complex is investigated by fluorescence recovery after photobleaching (FRAP) technique. Furthermore, cLSM image stacks of living cells are generated for the modeling of three dimensional (3-D) cell geometries. In order to prevent photochemical damage of the living cells induced by UV excitation of B[a]P, visualization is done by B[a]P’s auto fluorescence using near infrared two-photon-excitation. Murine Hepatoma 1c1c7 cells are exposed to graded concentrations of B[a]P (50 nM to 20 μM) for different incubation time periods (15 minutes to 48 hours). The highest amounts of B[a]P were found in lipid droplets and lysosomes, where the B[a]P molecules are collected and form large aggregates. We were able to work with concentrations down to 50 nM corresponding to that used for genomic and proteomic investigations. Also, for the first time imaging of B[a]P metabolites inside lipid droplets is presented in this work. The data and the model developed in this study will provide new insights into the systematic regulation of the B[a]P, the AhR as well as the receptor-ligand-complex pathway and the study will also serve as a prototype for elucidating other stress response pathways in the future.
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Mickler, Frauke Martina [Verfasser], and CHRISTOPH [Akademischer Betreuer] BRAEUCHLE. "Live-cell imaging elucidates cellular interactions of gene nanocarriers for cancer therapy / Frauke Martina Mickler. Betreuer: Christoph Bräuchle." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2013. http://d-nb.info/1047300516/34.
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Dittrich, Florian [Verfasser], and Stefan [Akademischer Betreuer] Landgraeber. "Untersuchungen zum abriebpartikelinduzierten Zelltod mittels "live-cell Imaging" im Kontext der aseptischen Endoprothesenlockerung / Florian Dittrich ; Betreuer: Stefan Landgraeber." Duisburg, 2018. http://d-nb.info/1155097262/34.
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Jensen, Rebecca Leah. "Live Cell Imaging to Investigate Bone Marrow Stromal Cell Adhesion and Migration on Titanium Surfaces: A Micro-Incubator in vitro Model." Cleveland State University / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=csu1391128419.
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Dursun, Ezgi [Verfasser], Anne [Akademischer Betreuer] Krug, Thomas [Gutachter] Korn, and Markus [Gutachter] Gerhard. "Cell fate decisions of common dendritic cell progenitors characterized by continuous live cell imaging at the single cell level / Ezgi Dursun ; Gutachter: Thomas Korn, Markus Gerhard ; Betreuer: Anne Krug." München : Universitätsbibliothek der TU München, 2015. http://d-nb.info/1114393983/34.
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Wen, Mary Mei. "New strategies for tagging quantum dots for dynamic cellular imaging." Diss., Georgia Institute of Technology, 2013. http://hdl.handle.net/1853/52150.
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In recent years, semiconductor quantum dots (QDs) have arisen as a new class of fluorescent probes that possess unique optical and electronic properties well-suited for single-molecule imaging of dynamic live cell processes. Nonetheless, the large size of conventional QD-ligand constructs has precluded their widespread use in single-molecule studies, especially on cell interiors. A typical QD-ligand construct can range upwards of 35 nm in diameter, well exceeding the size threshold for cytosolic diffusion and posing steric hindrance to binding cell receptors. The objective of this research is to develop tagging strategies that allow QD-ligand conjugates to specifically bind their target proteins while maintaining a small overall construct size. To achieve this objective, we utilize the HaloTag protein (HTP) available from Promega Corporation, which reacts readily with a HaloTag ligand (HTL) to form a covalent bond. When HaloTag ligands are conjugated to size-minimized multidentate polymer coated QDs, compact QD-ligand constructs less than 15 nm in diameter can be produced. These quantum dot-HaloTag ligand (QD-HTL) conjugates can then be used to covalently bind and track cellular receptors genetically fused to the HaloTag protein. In this study, size-minimized quantum dot-HaloTag ligand conjugates are synthesized and evaluated for their ability to bind specifically to purified and cellular HTP. The effect of QD-HTL surface modifications on different types of specific and nonspecific cellular binding are systematically investigated. Finally, these QD-HTL conjugates are utilized for single-molecule imaging of dynamic live cell processes. Our results show that size-minimized QD-HTLs exhibit great promise as novel imaging probes for live cell imaging, allowing researchers to visualize cellular protein dynamics in remarkable detail.
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Helfrich, Stefan Verfasser], Wolfgang [Akademischer Betreuer] Wiechert, and Björn [Akademischer Betreuer] [Usadel. "High-throughput live-cell imaging for investigations of cellular heterogeneity in Corynebacterium glutamicum / Stefan Helfrich ; Wolfgang Wiechert, Björn Usadel." Aachen : Universitätsbibliothek der RWTH Aachen, 2016. http://d-nb.info/1129875989/34.
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Boni, Andrea [Verfasser], and Jan [Akademischer Betreuer] Ellenberg. "Inner nuclear membrane protein targeting studied by quantitative live cell imaging and RNAi screening / Andrea Boni ; Betreuer: Jan Ellenberg." Heidelberg : Universitätsbibliothek Heidelberg, 2016. http://d-nb.info/1180608046/34.
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