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Manipulation and imaging of interactions between layer-by-layer capsules and live cells using nanopipettes and SICMChen, Yuxiu January 2018 (has links)
Usability of many chemical substances with significant potential for biomedical applications is limited by their poor solubility in water or limited stability in the physiological environment. One of promising strategies for therapeutic targeted delivery of these types of substances into cells and tissues is their encapsulation inside polyelectrolyte microcapsules (Volodkin et al., 2004b, Sukhorukov et al., 1998b). Successful internalisation of microcapsules loaded with various macromolecules have been observed in several types of living cells (Javier et al., 2008, Kastl et al., 2013), however the mechanisms of the uptake of capsules by living cells are not yet fully understood. Detailed understanding of physico-chemical and mechanical interactions between capsules and living cells is required for specific targeting, effective delivery, and elimination of any potential toxic side effects. This has been largely limited by capabilities of available imaging techniques and lack of specific fluorescent markers for certain types of cellular uptake. The rate of internalisation of microcapsules was primarily studied at the level of cell population using conventional optical/fluorescence microscopy, confocal microscopy, and flow cytometry (Gao et al., 2016, Ai et al., 2005, Sun et al., 2015). These conventional fluorescence methods are known to be prone to overestimating the number of internalised capsules due to their limited capability to exclude capsules which were not fully internalised and remained attached to the cell surface (Javier et al., 2006). Experimental evidence with resolution high enough to resolve the fine membrane processes interacting with microcapsules has been limited to fixed samples imaged by scanning electron microscopy and transmission electron microscopy (Kastl et al., 2013) capturing randomly timed "snapshots" of what is likely to be highly dynamic and complex interaction. Physical force interactions between cellular membrane and capsules during the internalisation were suggested to cause buckling of capsules based on indirect evidence obtained using fluorescence microscopy in live cells 15 (Palankar et al., 2013) and separate measurements of capsule deformation under colloidal probe atomic force microscopy (AFM) outside of the cellular environment (Delcea et al., 2010, Dubreuil et al., 2003). However, our knowledge of the mechanical properties of the fine membrane structures directly involved in the internalisation process or how these structures form during the internalisation is very limited, if non-existent. Here we employ a different approach based on a high-resolution scanning probe technique called scanning ion conductance microscopy (SICM). SICM uses reduction in ionic current through the probe represented by an electrolyte-filled glass nanopipette immersed in saline solution to detect proximity of sample surface (Hansma et al., 1989, Korchev et al., 1997a). The technique has been previously used for high-resolution scanning of biological samples of complexity similar to what can be expected in case of microcapsules interacting with cells (Novak et al., 2014, Novak et al., 2009), and also for mapping mechanical properties at high resolution (Ossola et al., 2015, Rheinlaender and Schaffer, 2013). It has been proved to be able to visualise internalisation process of 200 nm carboxy-modified latex nanoparticles (Novak et al., 2014), however it is not clear whether it would be suitable for visualising internalisation of substantially larger, microscale-sized particles. The aim of this research was to visualize the live internalisation process of microcapsules entering cells by using SICM. The first two chapters of this thesis are introduction and literature review, which summarise the current state of the art. Chapter 3 states the aim and objectives of this study. Chapter 4 introduces the materials and methods we used in our research. Chapter 5, 6, 7 present the main findings of our research. Chapter 5 states the challenges we met in visualising the live internalisation of microcapsule as well as our solution for overcoming those challenges. At the end of that chapter, we describe the detailed procedure we used for recording the live internalisation of microcapsules. The results we got using this procedure are presented in chapter 6 and 7. In chapter 8, we discuss the results we found by comparing them to the results of previous research. In chapter 9, we summarise our study and give some suggestions on future work.
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Investigation of voltage- and light-sensitive ion channelsFromme, Ulrich 29 February 2016 (has links)
No description available.
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Mechanoelectrochemistry of electroactive polymers using shear-force based near-field microscopyVenkatesh, Vijay 01 October 2020 (has links)
No description available.
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A scanning ion conductance microscopy assay to investigate interactions between cell penetrating peptides and pore-suspending membranesSaßen, Christoph 22 October 2013 (has links)
Die Rasterionenleitfähigkeitsmikroskopie (scanning ion conductance microscopy, SICM) stellt eine kontaktfreie Methode zur Ermittlung sowohl der Topographie als auch lokalen Ionenleitfähigkeit einer Oberfläche dar. Besonders vorteilhaft ist die Vermeidung mechanischer Beeinflussung bei der Untersuchung flexibler Strukturen, z.B. Lipiddoppelschichten wie Zellen oder künstlich erzeugter Lipidmembranen. Porenüberspannende Membranen (pore-suspending membranes, PSMs) verbinden als ein Beispiel für Modellsysteme eine hohe Stabilität mit lateraler Mobilität und dem Vorhandensein wässriger Kompartimente ober- und unterhalb der Doppelschicht, wie sie auch in der Natur gefunden werden. Ein wichtiges Forschungsgebiet stellt die Untersuchung der Wechselwirkung von Peptiden, besonders zellpenetrierenden Peptiden (cell penetrating peptides, CPPs), mit Lipiden und anderen Membranbestandteilen dar. Häufig untersuchte Beispiele sind Melittin, Hauptbestandteil des Giftes der Honigbiene Apis mellifera, sowie Penetratin, dritte Helix der Antennapedia Homöodomäne von Drosophila melanogaster.
Generalisierte Protokolle zur Herstellung lösungsmittelfreier PSMs werden vorgestellt. Riesige unilamellare Vesikel (giant unilamellar vesicles, GUVs) unterschiedlicher Lipidzusammensetzung wurden hierzu auf porösem Siliziumnitrid (Si3N4), welches mit Cholesterylpolyethylenoxythiol (CPEO3, hydrophob) bzw. Mercaptoethanol (ME, hydrophil) funktionalisiert worden war, gespreitet. Verwendet wurden GUVs aus reinen Phosphatidylcholin (PC)-Lipiden sowie aus Mischungen von PC-Lipiden mit Cholesterol und PC-Lipiden mit Phosphatidylserin (PS)-Lipiden. Der Erfolg des Spreitvorgangs wurde durch Abbilden mittels konfokaler Rasterlasermikroskopie (confocal laser scanning microscopy, CLSM) und SICM verifiziert.
Der Hauptteil dieser Arbeit behandelte die Entwicklung und Anwendung CLSM- und SICM-basierter CPP-Titrationsassays zur Aufklärung des Einflusses der Substratfunktionalisierung und der Lipidzusammensetzung der Membranen auf die Wechselwirkung zwischen Melittin bzw. Penetratin und den Lipiddoppelschichten. CLSM-Experimente wurden mit Melittin auf allen zur Verfügung stehenden PSMs sowohl auf hydrophob als auch hydrophil funktiona-lisierten Substraten durchgeführt, während Penetratin auf den drei unterschiedlichen PSMs auf hydrophil funktionalisierten Substraten verwendet wurde. Ein Reißen der Membranen wurde im Fall hydrophil funktionalisierter Substrate für beide Peptide im Bereich von 1–3 µM beobachtet. Bei hydrophob funktionalisierten Substraten induzierte eine dreifach geringere Melittinkonzentration die Zerstörung der Membranen. Sowohl auf hydrophob als auch auf hydrophil funktionalisierten Substraten wurde bei einem Cholesterolanteil von 10% eine Erhöhung der zum Reißen notwendigen Melittinkonzentratin erhalten, während bei 20% PS-Anteil eine Verschiebung zu geringeren Konzentrationen evident wurde. SICM-Experimente wurden mit Melittin auf PC/Cholesterol-PSMs auf hydrophob und hydrophil funktionalisierten Substraten und mit reinen PC-PSMs auf hydrophil funktionalisierten Membranen durchgeführt. Es wurden keine signifikanten Konzentrationsunterschiede beobachtet; die gefundenen Konzentrationsbereiche jedoch stimmten mit denen der CLSM-Experimente überein. Darüberhinaus wurde vor dem Reißen der Membranen ein Ansteigen der Porentiefe gefunden, das mit einer erhöhten Membranpermeabilität korrespondiert.
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Fabricação, caracterização e aplicação de pontas de prova eletroquímicas multifuncionais para técnicas de microscopia de varredura de ponta de prova eletroquímica / Fabrication, characterization and applications of multifunctional probes for scanning electrochemical probe microscopy techniquesMeloni, Gabriel Negrão 17 November 2017 (has links)
Esta tese apresenta os achados e avanços obtidos na fabricação, caracterização e aplicação de pontas de prova eletroquímicas multifuncionais para a obtenção de informações eletroquímicas resolvidas no espaço em diversas superfícies/interfaces por meio de técnicas de microscopia de varredura de ponta de prova eletroquímica (SEPM, em inglês). Diferentes designs de pontas de prova multifuncionais foram investigados e, devido a natureza não convencional destas, novos métodos para fabricação e caracterização foram desenvolvidos. Os benefícios da utilização de pontas de prova multifuncionais para a obtenção de informações eletroquímicas resolvidas no espaço ficaram evidente durante a realização de experimentos \"prova de conceito\", onde a maior densidade de informação obtida permitiu o estudo de sistemas mais complexos e a aquisição de informações eletroquímicas livre de interferência topográfica mesmo em superfícies não planas. A hibridização de diferentes técnicas de microscopia de varredura de ponta de prova eletroquímica em uma única ponta de prova também foi investigada o que se provou extremamente útil para a aquisição de imagens eletroquímicas de alta resolução, livres de influências topográficas, quando utilizada a técnica de microscopia de condutividade iônica (SICM, em inglês) como sensor de topografia do substrato investigado. Por ultimo, uma nova técnica, baseada na microscopia de condutividade iônica, que se utiliza de pontas de prova eletroquímicas multifuncionais fabricadas a partir de uma nanopipeta de um único canal, foi desenvolvida. Esta nova técnica se mostrou extremamente ponderosa, capaz de obter informações a respeito da topografia e mapear sítios ativos sobre um substrato utilizando uma nanopipeta de um único canal com alta resolução especial e temporal a uma taxa de aproximadamente 4000 pixels por Segundo. / This thesis presents the findings and advances made on fabrication, characterization and application of multifunctional electrochemical probes to acquire space resolved electrochemical information on diverse surfaces/interfaces employing Scanning Electrochemical Probe Microscopy (SEPM) techniques. Different multifunctional probes designs were investigated and new and innovative methods for fabrication and characterization of those probes were developed, which was necessary due to the unconventional nature of most of the probes studied. The benefits of using multifunctional probes for space resolved electrochemical measurements was clear during \"proof-of-concept\" experiments, where the increased density of information allowed the study of complex systems and the acquisition of topography-free electrochemical information of rough surfaces. The hybridization of different SEPM techniques in a single probe tip was also investigated, and this was found to be extremely beneficial, especially for acquiring high-resolution, topography-free, electrochemical images employing Scanning Ion Conductance Microscopy as a topography feedback. Finally, a new SICM technique, based on the use of a multifunctional probe tip fabricated from a single barrel nanopipette, was developed. This new technique was found to be extremely powerful, capable of acquiring information on topography and map active sites over substrates using a single barrel pipette with high spatial and temporal resolution at a rate of approx. 4000 pixels per second.
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Fabricação, caracterização e aplicação de pontas de prova eletroquímicas multifuncionais para técnicas de microscopia de varredura de ponta de prova eletroquímica / Fabrication, characterization and applications of multifunctional probes for scanning electrochemical probe microscopy techniquesGabriel Negrão Meloni 17 November 2017 (has links)
Esta tese apresenta os achados e avanços obtidos na fabricação, caracterização e aplicação de pontas de prova eletroquímicas multifuncionais para a obtenção de informações eletroquímicas resolvidas no espaço em diversas superfícies/interfaces por meio de técnicas de microscopia de varredura de ponta de prova eletroquímica (SEPM, em inglês). Diferentes designs de pontas de prova multifuncionais foram investigados e, devido a natureza não convencional destas, novos métodos para fabricação e caracterização foram desenvolvidos. Os benefícios da utilização de pontas de prova multifuncionais para a obtenção de informações eletroquímicas resolvidas no espaço ficaram evidente durante a realização de experimentos \"prova de conceito\", onde a maior densidade de informação obtida permitiu o estudo de sistemas mais complexos e a aquisição de informações eletroquímicas livre de interferência topográfica mesmo em superfícies não planas. A hibridização de diferentes técnicas de microscopia de varredura de ponta de prova eletroquímica em uma única ponta de prova também foi investigada o que se provou extremamente útil para a aquisição de imagens eletroquímicas de alta resolução, livres de influências topográficas, quando utilizada a técnica de microscopia de condutividade iônica (SICM, em inglês) como sensor de topografia do substrato investigado. Por ultimo, uma nova técnica, baseada na microscopia de condutividade iônica, que se utiliza de pontas de prova eletroquímicas multifuncionais fabricadas a partir de uma nanopipeta de um único canal, foi desenvolvida. Esta nova técnica se mostrou extremamente ponderosa, capaz de obter informações a respeito da topografia e mapear sítios ativos sobre um substrato utilizando uma nanopipeta de um único canal com alta resolução especial e temporal a uma taxa de aproximadamente 4000 pixels por Segundo. / This thesis presents the findings and advances made on fabrication, characterization and application of multifunctional electrochemical probes to acquire space resolved electrochemical information on diverse surfaces/interfaces employing Scanning Electrochemical Probe Microscopy (SEPM) techniques. Different multifunctional probes designs were investigated and new and innovative methods for fabrication and characterization of those probes were developed, which was necessary due to the unconventional nature of most of the probes studied. The benefits of using multifunctional probes for space resolved electrochemical measurements was clear during \"proof-of-concept\" experiments, where the increased density of information allowed the study of complex systems and the acquisition of topography-free electrochemical information of rough surfaces. The hybridization of different SEPM techniques in a single probe tip was also investigated, and this was found to be extremely beneficial, especially for acquiring high-resolution, topography-free, electrochemical images employing Scanning Ion Conductance Microscopy as a topography feedback. Finally, a new SICM technique, based on the use of a multifunctional probe tip fabricated from a single barrel nanopipette, was developed. This new technique was found to be extremely powerful, capable of acquiring information on topography and map active sites over substrates using a single barrel pipette with high spatial and temporal resolution at a rate of approx. 4000 pixels per second.
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Localised dosing and nanodetection using a novel scanning ion conductance microscope and its application to Alzheimer's diseaseChen, Wei-Hsin Chen January 2018 (has links)
Scanning ion conductance microscopy (SICM) is a technique for non-contact topographic imaging. In this thesis, a biophysical investigation into Alzheimer's Disease (AD) was carried, with toxic oligomers dosed locally and quantitatively on to single astrocytes using SICM and simultaneously monitoring the response of the target cell. Examination of the effectiveness of antibodies that bind to Abeta or alpha-synuclein (Asyn)peptides depends on the measurement of oligomer-induced abnormal calcium homeostasis in single astrocytes. The method was shown to work at physiological concentrations of oligomers. A series of experiments measuring the reduction in calcium inux in mixtures of antibodies and cerebrospinal fluid (CSF) of AD patients suggested that the binding to co-oligomers composed of Abeta and Asyn may be crucial in the treatment of AD. Furthermore, it may be beneficial to test antibodies before the clinical trial using this assay. The mechanism of this entry of calcium is hypothesised to be the result of the formation of oligomer-induced transient pores in the cell membrane. To verify this hypothesis, a new SICM instrument was built with two nanopipettes; one for dosing and one for detection of the adenosine triphosphate (ATP) release from these pores. A variety of different ATP sensors were made. The best had a sensitivity of 10 micro molar and works as a hexokinase-cofunctioned electrolyte-gated organic field-effect-transistor. However no statistically significant results for ATP release have been obtained in the experiments performed to date. Overall this thesis describes new biophysical methods to study the effect of protein aggregates on live cells and the effectiveness of potential therapies, such as antibodies and nanobodies, to reduce these aggregate induced effects. It can be applied to synthetic aggregates of Abeta or the aggregates present in human CSF.
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