TY - GEN A1 - Zór, K. A1 - Heiskanen, A. A1 - Caviglia, Claudia A1 - Vergani, M. A1 - Landini, E. A1 - Shah, F. A1 - Carminati, Marco A1 - Martínez-Serrano, A. A1 - Ramos Moreno, T. A1 - Kokaia, M. A1 - Benayahu, Dafna A1 - Keresztes, Zs. A1 - Papkovsky, D. A1 - Wollenberger, Ursula A1 - Svendsen, W. E. A1 - Dimaki, M. A1 - Ferrari, G. A1 - Raiteri, R. A1 - Sampietro, M. A1 - Dufva, M. A1 - Emnéus, J. T1 - A compact multifunctional microfluidic platform for exploring cellular dynamics in real-time using electrochemical detection N2 - Downscaling of microfluidic cell culture and detection devices for electrochemical monitoring has mostly focused on miniaturization of the microfluidic chips which are often designed for specific applications and therefore lack functional flexibility. We present a compact microfluidic cell culture and electrochemical analysis platform with in-built fluid handling and detection, enabling complete cell based assays comprising on-line electrode cleaning, sterilization, surface functionalization, cell seeding, cultivation and electrochemical real-time monitoring of cellular dynamics. To demonstrate the versatility and multifunctionality of the platform, we explored amperometric monitoring of intracellular redox activity in yeast (Saccharomyces cerevisiae) and detection of exocytotically released dopamine from rat pheochromocytoma cells (PC12). Electrochemical impedance spectroscopy was used in both applications for monitoring cell sedimentation and adhesion as well as proliferation in the case of PC12 cells. The influence of flow rate on the signal amplitude in the detection of redox metabolism as well as the effect of mechanical stimulation on dopamine release were demonstrated using the programmable fluid handling capability. The here presented platform is aimed at applications utilizing cell based assays, ranging from e.g. monitoring of drug effects in pharmacological studies, characterization of neural stem cell differentiation, and screening of genetically modified microorganisms to environmental monitoring. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 289 Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-99492 ER - TY - GEN A1 - Wolff, Martin A1 - Gast, Klaus A1 - Evers, Andreas A1 - Kurz, Michael A1 - Pfeiffer-Marek, Stefania A1 - Schüler, Anja A1 - Seckler, Robert A1 - Thalhammer, Anja T1 - A Conserved Hydrophobic Moiety and Helix-Helix Interactions Drive the Self-Assembly of the Incretin Analog Exendin-4 T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Exendin-4 is a pharmaceutical peptide used in the control of insulin secretion. Structural information on exendin-4 and related peptides especially on the level of quaternary structure is scarce. We present the first published association equilibria of exendin-4 directly measured by static and dynamic light scattering. We show that exendin-4 oligomerization is pH dependent and that these oligomers are of low compactness. We relate our experimental results to a structural hypothesis to describe molecular details of exendin-4 oligomers. Discussion of the validity of this hypothesis is based on NMR, circular dichroism and fluorescence spectroscopy, and light scattering data on exendin-4 and a set of exendin-4 derived peptides. The essential forces driving oligomerization of exendin-4 are helix–helix interactions and interactions of a conserved hydrophobic moiety. Our structural hypothesis suggests that key interactions of exendin-4 monomers in the experimentally supported trimer take place between a defined helical segment and a hydrophobic triangle constituted by the Phe22 residues of the three monomeric subunits. Our data rationalize that Val19 might function as an anchor in the N-terminus of the interacting helix-region and that Trp25 is partially shielded in the oligomer by C-terminal amino acids of the same monomer. Our structural hypothesis suggests that the Trp25 residues do not interact with each other, but with C-terminal Pro residues of their own monomers. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1161 KW - biophysics KW - diabetes KW - peptides KW - oligomerization KW - conformational change KW - molecular modeling KW - static and dynamic light scattering KW - spectroscopy Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-522081 SN - 1866-8372 IS - 9 ER - TY - GEN A1 - Liaimer, Anton A1 - Jensen, John B. A1 - Dittmann-Thünemann, Elke T1 - A genetic and chemical perspective on symbiotic recruitment of cyanobacteria of the genus Nostoc into the host plant Blasia pusilla L. T2 - Frontiers in microbiology N2 - Liverwort Blasia pusilla L. recruits soil nitrogen-fixing cyanobacteria of genus Nostoc as symbiotic partners. In this work we compared Nostoc community composition inside the plants and in the soil around them from two distant locations in Northern Norway. STRR fingerprinting and 16S rDNA phylogeny reconstruction showed a remarkable local diversity among isolates assigned to several Nostoc clades. An extensive web of negative allelopathic interactions was recorded at an agricultural site, but not at the undisturbed natural site. The cell extracts of the cyanobacteria did not show antimicrobial activities, but four isolates were shown to be cytotoxic to human cells. The secondary metabolite profiles of the isolates were mapped by MALDI-TOF MS, and the most prominent ions were further analyzed by Q-TOF for MS/MS aided identification. Symbiotic isolates produced a great variety of small peptide-like substances, most of which lack any record in the databases. Among identified compounds we found microcystin and nodularin variants toxic to eukaryotic cells. Microcystin producing chemotypes were dominating as symbiotic recruits but not in the free-living community. In addition, we were able to identify several novel aeruginosins and banyaside-like compounds, as well as nostocyclopeptides and nosperin. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 434 KW - cyanobacteria KW - secondary metabolites KW - symbiosis KW - Blasia KW - Nostoc KW - allelopathy Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-407179 ER - TY - GEN A1 - Zhang, Youjun A1 - Chen, Moxian A1 - Siemiatkowska, Beata A1 - Toleco, Mitchell Rey A1 - Jing, Yue A1 - Strotmann, Vivien A1 - Zhang, Jianghua A1 - Stahl, Yvonne A1 - Fernie, Alisdair R. T1 - A highly efficient agrobacterium-mediated method for transient gene expression and functional studies in multiple plant species T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Although the use of stable transformation technology has led to great insight into gene function, its application in high-throughput studies remains arduous. Agro-infiltration have been widely used in species such as Nicotiana benthamiana for the rapid detection of gene expression and protein interaction analysis, but this technique does not work efficiently in other plant species, including Arabidopsis thaliana. As an efficient high-throughput transient expression system is currently lacking in the model plant species A. thaliana, we developed a method that is characterized by high efficiency, reproducibility, and suitability for transient expression of a variety of functional proteins in A. thaliana and 7 other plant species, including Brassica oleracea, Capsella rubella, Thellungiella salsuginea, Thellungiella halophila, Solanum tuberosum, Capsicum annuum, and N. benthamiana. Efficiency of this method was independently verified in three independent research facilities, pointing to the robustness of this technique. Furthermore, in addition to demonstrating the utility of this technique in a range of species, we also present a case study employing this method to assess protein-protein interactions in the sucrose biosynthesis pathway in Arabidopsis. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1189 KW - transient expression KW - agro-infiltration KW - subcellular localization KW - protein-protein interaction Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-524254 SN - 1866-8372 IS - 5 ER - TY - GEN A1 - Dortay, Hakan A1 - Müller-Röber, Bernd T1 - A highly efficient pipeline for protein expression in Leishmania tarentolae sing infrared fluorescence protein as marker N2 - Background: Leishmania tarentolae, a unicellular eukaryotic protozoan, has been established as a novel host for recombinant protein production in recent years. Current protocols for protein expression in Leishmania are, however, time consuming and require extensive lab work in order to identify well-expressing cell lines. Here we established an alternative protein expression work-flow that employs recently engineered infrared fluorescence protein (IFP) as a suitable and easy-to-handle reporter protein for recombinant protein expression in Leishmania. As model proteins we tested three proteins from the plant Arabidopsis thaliana, including a NAC and a type-B ARR transcription factor. Results: IFP and IFP fusion proteins were expressed in Leishmania and rapidly detected in cells by deconvolution microscopy and in culture by infrared imaging of 96-well microtiter plates using small cell culture volumes (2 μL - 100 μL). Motility, shape and growth of Leishmania cells were not impaired by intracellular accumulation of IFP. In-cell detection of IFP and IFP fusion proteins was straightforward already at the beginning of the expression pipeline and thus allowed early pre-selection of well-expressing Leishmania clones. Furthermore, IFP fusion proteins retained infrared fluorescence after electrophoresis in denaturing SDS-polyacrylamide gels, allowing direct in-gel detection without the need to disassemble cast protein gels. Thus, parameters for scaling up protein production and streamlining purification routes can be easily optimized when employing IFP as reporter. Conclusions: Using IFP as biosensor we devised a protocol for rapid and convenient protein expression in Leishmania tarentolae. Our expression pipeline is superior to previously established methods in that it significantly reduces the hands-on-time and work load required for identifying well-expressing clones, refining protein production parameters and establishing purification protocols. The facile in-cell and in-gel detection tools built on IFP make Leishmania amenable for high-throughput expression of proteins from plant and animal sources. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 122 KW - System KW - Donovani Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-44773 ER - TY - GEN A1 - Dortay, Hakan A1 - Müller-Röber, Bernd T1 - A highly efficient pipeline for protein expression in Leishmania tarentolae using infrared fluorescence protein as marker N2 - Background: Leishmania tarentolae, a unicellular eukaryotic protozoan, has been established as a novel host for recombinant protein production in recent years. Current protocols for protein expression in Leishmania are, however, time consuming and require extensive lab work in order to identify well-expressing cell lines. Here we established an alternative protein expression work-flow that employs recently engineered infrared fluorescence protein (IFP) as a suitable and easy-to-handle reporter protein for recombinant protein expression in Leishmania. As model proteins we tested three proteins from the plant Arabidopsis thaliana, including a NAC and a type-B ARR transcription factor. Results: IFP and IFP fusion proteins were expressed in Leishmania and rapidly detected in cells by deconvolution microscopy and in culture by infrared imaging of 96-well microtiter plates using small cell culture volumes (2 mu L - 100 mu L). Motility, shape and growth of Leishmania cells were not impaired by intracellular accumulation of IFP. In-cell detection of IFP and IFP fusion proteins was straightforward already at the beginning of the expression pipeline and thus allowed early pre-selection of well-expressing Leishmania clones. Furthermore, IFP fusion proteins retained infrared fluorescence after electrophoresis in denaturing SDS-polyacrylamide gels, allowing direct in-gel detection without the need to disassemble cast protein gels. Thus, parameters for scaling up protein production and streamlining purification routes can be easily optimized when employing IFP as reporter. Conclusions: Using IFP as biosensor we devised a protocol for rapid and convenient protein expression in Leishmania tarentolae. Our expression pipeline is superior to previously established methods in that it significantly reduces the hands-on-time and work load required for identifying well-expressing clones, refining protein production parameters and establishing purification protocols. The facile in-cell and in-gel detection tools built on IFP make Leishmania amenable for high-throughput expression of proteins from plant and animal sources. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 366 KW - System KW - Donovani Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-400876 ER - TY - GEN A1 - Jantzen, Friederike A1 - Wozniak, Natalia Joanna A1 - Kappel, Christian A1 - Sicard, Adrien A1 - Lenhard, Michael T1 - A high‑throughput amplicon‑based method for estimating outcrossing rates T2 - Postprints der Universität Potsdam Mathematisch-Naturwissenschaftliche Reihe N2 - Background: The outcrossing rate is a key determinant of the population-genetic structure of species and their long-term evolutionary trajectories. However, determining the outcrossing rate using current methods based on PCRgenotyping individual offspring of focal plants for multiple polymorphic markers is laborious and time-consuming. Results: We have developed an amplicon-based, high-throughput enabled method for estimating the outcrossing rate and have applied this to an example of scented versus non-scented Capsella (Shepherd’s Purse) genotypes. Our results show that the method is able to robustly capture differences in outcrossing rates. They also highlight potential biases in the estimates resulting from differential haplotype sharing of the focal plants with the pollen-donor population at individual amplicons. Conclusions: This novel method for estimating outcrossing rates will allow determining this key population-genetic parameter with high-throughput across many genotypes in a population, enabling studies into the genetic determinants of successful pollinator attraction and outcrossing. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 745 KW - Outcrossing KW - Mixed mating KW - Outcrossing rate KW - Capsella KW - Amplicon sequencing Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-435657 SN - 1866-8372 IS - 745 ER - TY - GEN A1 - Cox, Tom A1 - Maris, Tom A1 - Soetart, Karline A1 - Conley, Daniel A1 - van Damme, Stefan A1 - Meire, Patrick A1 - Middelburg, Jack J. A1 - Vos, Matthijs A1 - Struyf, Eric T1 - A macro-tidal freshwater ecosystem recovering from hypereutrophication : the Schelde lease study N2 - We report a 40 year record of eutrophication and hypoxia on an estuarine ecosystem and its recovery from hypereutrophication. After decades of high inorganic nutrient concentrations and recurring anoxia and hypoxia, we observe a paradoxical increase in chlorophyll-a concentrations with decreasing nutrient inputs. We hypothesise that algal growth was inhibited due to hypereutrophication, either by elevated ammonium concentrations, severe hypoxia or the production of harmful substances in such a reduced environment. We study the dynamics of a simple but realistic mathematical model, incorporating the assumption of algal growth inhibition. It shows a high algal biomass, net oxygen production equilibrium with low ammonia inputs, and a low algal biomass, net oxygen consumption equilibrium with high ammonia inputs. At intermediate ammonia inputs it displays two alternative stable states. Although not intentional, the numerical output of this model corresponds to observations, giving extra support for assumption of algal growth inhibition. Due to potential algal growth inhibition, the recovery of hypereutrophied systems towards a classical eutrophied state, will need reduction of waste loads below certain thresholds and will be accompanied by large fluctuations in oxygen concentrations. We conclude that also flow-through systems, heavily influenced by external forcings which partly mask internal system dynamics, can display multiple stable states. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 144 KW - Estuary SW Netherlands KW - southern North-Sea KW - Past 50 years KW - Westerschelde estuary KW - Marine ecosystems Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-45180 ER - TY - THES A1 - Kirschbaum, Michael T1 - A microfluidic approach for the initiation and investigation of surface-mediated signal transduction processes on a single-cell level T1 - Entwicklung einer mikrofluidischen Prozesslinie für die Induktion und Analyse oberflächenvermittelter Signaltransduktionsprozesse auf Einzelzell-Ebene N2 - For the elucidation of the dynamics of signal transduction processes that are induced by cellular interactions, defined events along the signal transduction cascade and subsequent activation steps have to be analyzed and then also correlated with each other. This cannot be achieved by ensemble measurements because averaging biological data ignores the variability in timing and response patterns of individual cells and leads to highly blurred results. Instead, only a multi-parameter analysis at a single-cell level is able to exploit the information that is crucially needed for deducing the signaling pathways involved. The aim of this work was to develop a process line that allows the initiation of cell-cell or cell-particle interactions while at the same time the induced cellular reactions can be analyzed at various stages along the signal transduction cascade and correlated with each other. As this approach requires the gentle management of individually addressable cells, a dielectrophoresis (DEP)-based microfluidic system was employed that provides the manipulation of microscale objects with very high spatiotemporal precision and without the need of contacting the cell membrane. The system offers a high potential for automation and parallelization. This is essential for achieving a high level of robustness and reproducibility, which are key requirements in order to qualify this approach for a biomedical application. As an example process for intercellular communication, T cell activation has been chosen. The activation of the single T cells was triggered by contacting them individually with microbeads that were coated with antibodies directed against specific cell surface proteins, like the T cell receptor-associated kinase CD3 and the costimulatory molecule CD28 (CD; cluster of differentiation). The stimulation of the cells with the functionalized beads led to a rapid rise of their cytosolic Ca2+ concentration which was analyzed by a dual-wavelength ratiometric fluorescence measurement of the Ca2+-sensitive dye Fura-2. After Ca2+ imaging, the cells were isolated individually from the microfluidic system and cultivated further. Cell division and expression of the marker molecule CD69 as a late activation event of great significance were analyzed the following day and correlated with the previously recorded Ca2+ traces for each individual cell. It turned out such that the temporal profile of the Ca2+ traces between both activated and non-activated cells as well as dividing and non-dividing cells differed significantly. This shows that the pattern of Ca2+ signals in T cells can provide early information about a later reaction of the cell. As isolated cells are highly delicate objects, a precondition for these experiments was the successful adaptation of the system to maintain the vitality of single cells during and after manipulation. In this context, the influences of the microfluidic environment as well as the applied electric fields on the vitality of the cells and the cytosolic Ca2+ concentration as crucially important physiological parameters were thoroughly investigated. While a short-term DEP manipulation did not affect the vitality of the cells, they showed irregular Ca2+ transients upon exposure to the DEP field only. The rate and the strength of these Ca2+ signals depended on exposure time, electric field strength and field frequency. By minimizing their occurrence rate, experimental conditions were identified that caused the least interference with the physiology of the cell. The possibility to precisely control the exact time point of stimulus application, to simultaneously analyze short-term reactions and to correlate them with later events of the signal transduction cascade on the level of individual cells makes this approach unique among previously described applications and offers new possibilities to unravel the mechanisms underlying intercellular communication. N2 - Zelluläre Interaktionen sind wirkungsvolle Mechanismen zur Kontrolle zellulärer Zustände in vivo. Für die Entschlüsselung der dabei beteiligten Signaltransduktionsprozesse müssen definierte Ereignisse entlang der zellulären Signalkaskade erfasst und ihre wechselseitige Beziehung zueinander aufgeklärt werden. Dies kann von Ensemble-Messungen nicht geleistet werden, da die Mittelung biologischer Daten die Variabilität des Antwortverhaltens individueller Zellen missachtet und verschwommene Resultate liefert. Nur eine Multiparameteranalyse auf Einzelzellebene kann die entscheidenden Informationen liefern, die für ein detailliertes Verständnis zellulärer Signalwege unabdingbar sind. Ziel der vorliegenden Arbeit war die Entwicklung einer Methode, welche die gezielte Kontaktierung einzelner Zellen mit anderen Zellen oder Partikeln ermöglicht und mit der die dadurch ausgelösten zellulären Reaktionen auf unterschiedlichen zeitlichen Ebenen analysiert und miteinander korreliert werden können. Da dies die schonende Handhabung einzeln adressierbarer Zellen erfordert, wurde ein auf Dielektrophorese (DEP) basierendes mikrofluidisches System eingesetzt, welches die berührungslose Manipulation mikroskaliger Objekte mit hoher zeitlicher und örtlicher Präzision erlaubt. Das System besitzt ein hohes Potential zur Automatisierung und Parallelisierung, was für eine robuste und reproduzierbare Analyse lebender Zellen essentiell, und daher eine wichtige Voraussetzung für eine Anwendung in der Biomedizin ist. Als Modellsystem für interzelluläre Kommunikation wurde die T-Zell-Aktivierung gewählt. Die Aktivierung der einzelnen T-Zellen wurde durch ihre gezielte Kontaktierung mit Mikropartikeln („beads“) induziert, welche mit Antikörpern gegen spezielle Oberflächenproteine, wie die dem T-Zell-Rezeptor assoziierte Kinase CD3 oder das kostimulatorische Protein CD28, beschichtet waren. Die Stimulation der Zellen mit den funktionalisierten beads führte zu einem raschen Anstieg der intrazellulären Ca2+-Konzentration, welche über eine ratiometrische Detektion des Ca2+-sensitiven Fluoreszenzfarbstoffs Fura-2 gemessen wurde. Anschließend wurden die einzelnen Zellen aus dem mikrofluidischen System isoliert und weiterkultiviert. Am nächsten Tag wurden Zellteilung und die CD69-Expression – ein wichtiger Marker für aktivierte T-Zellen – analysiert und auf Ebene der individuellen Zelle mit dem zuvor gemessenen Ca2+-Signal korreliert. Es stellte sich heraus, dass der zeitliche Verlauf des intrazellulären Ca2+-Signals zwischen aktivierten und nicht aktivierten, sowie zwischen geteilten und nicht geteilten Zellen signifikant verschieden war. Dies zeigt, dass Ca2+-Signale in stimulierten T-Zellen wichtige Informationen über eine spätere Reaktion der Zelle liefern können. Da Einzelzellen äußerst empfindlich auf ihre Umgebungsbedingungen reagieren, war die Anpassung der experimentellen Vorgehensweise im Hinblick auf die Zellverträglichkeit von großer Bedeutung. Vor diesem Hintergrund wurde der Einfluss sowohl der mikrofluidischen Umgebung, als auch der elektrischen Felder auf die Überlebensrate und die intrazelluläre Ca2+-Konzentration der Zellen untersucht. Während eine kurzzeitige DEP-Manipulation im mikrofluidischen System die Vitalität der Zellen nicht beeinträchtigte, zeigten diese unregelmäßige Fluktuationen ihrer intrazellulären Ca2+-Konzentration selbst bei geringer elektrischer Feldexposition. Die Ausprägung dieser Fluktuationen war abhängig von der Expositionszeit, der elektrischen Feldstärke und der Feldfrequenz. Über die Minimierung ihres Auftretens konnten experimentelle Bedingungen mit dem geringsten Einfluss auf die Physiologie der Zellen identifiziert werden. Die Möglichkeit, einzelne Zellen zeitlich definiert und präzise mit anderen Zellen oder Oberflächen zu kontaktieren, die unmittelbare Reaktion der Zellen zu messen und diese mit späteren Ereignissen der Zellantwort zu korrelieren, macht die hier vorgestellte Methode einzigartig im Vergleich mit anderen Ansätzen und eröffnet neue Wege, die der interzellulären Kommunikation zugrunde liegenden Mechanismen aufzuklären. KW - T-Zell Aktivierung KW - Calcium KW - Einzelzellen KW - Mikrofluidik KW - Dielektrophorese KW - T cell activation KW - calcium KW - single-cell KW - lab on a chip KW - dielectrophoresis Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-39576 ER - TY - THES A1 - Gerling, Marten Tobias T1 - A microfluidic system for high-precision image-based live cell sorting using dielectrophoretic forces T1 - Ein mikrofluidisches System zur hochpräzisen und bildgestützten Sortierung lebender Zellen mittels dielektrophoretischer Kräfte N2 - An important goal in biotechnology and (bio-) medical research is the isolation of single cells from a heterogeneous cell population. These specialised cells are of great interest for bioproduction, diagnostics, drug development, (cancer) therapy and research. To tackle emerging questions, an ever finer differentiation between target cells and non-target cells is required. This precise differentiation is a challenge for a growing number of available methods. Since the physiological properties of the cells are closely linked to their morphology, it is beneficial to include their appearance in the sorting decision. For established methods, this represents a non addressable parameter, requiring new methods for the identification and isolation of target cells. Consequently, a variety of new flow-based methods have been developed and presented in recent years utilising 2D imaging data to identify target cells within a sample. As these methods aim for high throughput, the devices developed typically require highly complex fluid handling techniques, making them expensive while offering limited image quality. In this work, a new continuous flow system for image-based cell sorting was developed that uses dielectrophoresis to precisely handle cells in a microchannel. Dielectrophoretic forces are exerted by inhomogeneous alternating electric fields on polarisable particles (here: cells). In the present system, the electric fields can be switched on and off precisely and quickly by a signal generator. In addition to the resulting simple and effective cell handling, the system is characterised by the outstanding quality of the image data generated and its compatibility with standard microscopes. These aspects result in low complexity, making it both affordable and user-friendly. With the developed cell sorting system, cells could be sorted reliably and efficiently according to their cytosolic staining as well as morphological properties at different optical magnifications. The achieved purity of the target cell population was up to 95% and about 85% of the sorted cells could be recovered from the system. Good agreement was achieved between the results obtained and theoretical considerations. The achieved throughput of the system was up to 12,000 cells per hour. Cell viability studies indicated a high biocompatibility of the system. The results presented demonstrate the potential of image-based cell sorting using dielectrophoresis. The outstanding image quality and highly precise yet gentle handling of the cells set the system apart from other technologies. This results in enormous potential for processing valuable and sensitive cell samples. N2 - Ein wichtiges Ziel in der Biotechnologie und (bio-) medizinischen Forschung ist die Isolation von Einzelzellen aus einer heterogenen Zellpopulation. Aufgrund ihrer besonderen Eigenschaften sind diese Zellen für die Bioproduktion, Diagnostik, Arzneimittelentwicklung, (Krebs-) Therapie und Forschung von großem Interesse. Um neue Fragestellungen angehen zu können, ist eine immer feinere Differenzierung zwischen Zielzellen und Nicht-Zielzellen erforderlich. Diese präzise Differenzierung stellt eine Herausforderung für eine wachsende Zahl verfügbarer Methoden dar. Da die physiologischen Eigenschaften der Zellen eng mit ihrer Morphologie verknüpft sind, ist es sinnvoll ihr Erscheinungsbild mit in die Sortierentscheidung einzubeziehen. Für etablierte Methoden stellt dies jedoch einen nicht adressierbaren Parameter dar, weshalb neue Methoden zur Identifikation und Isolation der Zielzellen benötigt werden. Folglich wurde in den letzten Jahren eine Vielzahl neuer durchflussbasierter Methoden entwickelt und vorgestellt, die 2D-Bilddaten zur Identifikation der Zielzellen innerhalb einer Probe heranziehen. Da diese Methoden auf hohe Durchsätze abzielen, erfordern die entwickelten Geräte in der Regel hochkomplexe Techniken zur Handhabung der Flüssigkeiten, was sie teuer macht und gleichzeitig zu einer begrenzten Bildqualität führt. In dieser Arbeit wurde ein neues durchflussbasiertes System zur bildbasierten Zellsortierung entwickelt, das Dielektrophorese zur präzisen Handhabung der Zellen in einem Mikrokanal verwendet. Dielektrophoretische Kräfte werden von inhomogenen elektrischen Wechselfeldern auf polarisierbare Partikel (hier: Zellen) ausgeübt. Bei dem vorliegenden System können die elektrischen Felder durch einen Signalgenerator präzise und schnell ein- und ausgeschaltet werden. Neben der daraus resultierenden einfachen und effektiven Handhabung der Zellen zeichnet sich das System durch die hervorragende Qualität der erzeugten Bilddaten und die Kompatibilität mit Standardmikroskopen aus. Diese Aspekte führen zu einer geringen Komplexität, die es sowohl erschwinglich als auch benutzerfreundlich macht. Mit dem entwickelten System zur Zellsortierung konnten Zellen sowohl auf Basis einer zytosolischen Färbung als auch auf Basis morphologischer Eigenschaften bei verschiedenen optischen Vergrößerungen zuverlässig und effizient sortiert werden. Die erreichte Reinheit der Zielzellpopulation betrug bis zu 95% wobei etwa 85% der sortierten Zellen aus dem System zurückgewonnen werden konnten. Dabei wurde eine gute Übereinstimmung der ermittelten Ergebnisse mit theoretischen Überlegungen erreicht. Der erreichte Durchsatz des Systems betrug bis zu 12.000 Zellen pro Stunde. Untersuchungen der Zellvitalität deuteten darauf hin, dass das System eine hohe Biokompatibilität aufweist. Die vorgestellten Ergebnisse demonstrieren das Potenzial der bildbasierten Zellsortierung mittels Dielektrophorese. Die herausragende Bildqualität und hochpräzise aber dennoch zellschonende Handhabung der Zellen grenzen das System von anderen Technologien ab. Daraus ergibt sich ein enormes Potenzial für die Verarbeitung wertvoller und sensibler Zellproben. KW - microfluidics KW - cell sorting KW - dielectrophoresis KW - Zellsortierung KW - Dielektrophorese KW - Mikrofluidik Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-587421 ER -