Imaging and Spectroscopic Mapping of Blood Cell Activity : Nanoparticles and Neutrophil Extracellular Traps

Sammanfattning: Imaging and visualization of cell activity when exposed to nanomaterial are of main importance, when investigating biological response to a wide range of biomaterials from medical implants to smart nanoprobes. The ability to provide molecular and chemical information with spatial resolution in the region of sub-µm leads to increased insight and understanding of these biological challenges. Interdisciplinary collaborative effort may contribute and help solving urgent matters related to the challenges that we globally share. It is necessary to develop powerful tools such as analytical imaging techniques for addressing these urgent issues. This will increase our knowledge from the visualization on the cellular and subcellular level and help designing sustainable, personalized medical nanoprobes. In this thesis, the focus is to investigate the possibilities using the fluorescence microscopy, combined with surface analytical techniques delivering element specific information. Neutrophils are the most abundant immune cell in our bodies. They scavenge the body for threats and are usually among the first ones to find intruders and start the inflammation process. They have several ways of handling a threat, the main three being degranulation, phagocytosis, and neutrophil extracellular traps (NETs). In short, degranulation where granules are released into the extracellular matrix, phagocytosis is the process when for example the bacteria in engulfed by the neutrophil and neutralized. The NETs are when the neutrophil decondense their DNA and throw it out as a net to physically trap the invader and together with reactive oxygen species, proteases, and other antimicrobial molecules. It has been observed that nanoparticles (NP) can trigger NETs and there have been some comparisons between different parameters such as size, geometry, and functionalization. In this thesis we have explored how to measure neutrophil activity by a novel label free and noninvasive method (Paper 1). The NanoEsca, a combined XPS and PEEM instrument, is used to chemically map the neutrophils and NETs. We could clearly observe the NETs in PEEM and XPS mode. Quantum Dots (QDots, CdSe based) was used to trigger NETs. We track down the Quantum Dots with the element specific mapping. In the next paper we further explored how to extract new information with this advanced instrument that is traditionally is used for material- and surface science, and just recently deliver results in imaging and visualization within life sciences. Ultrathin slices of neutrophils where made special focus was given to the research work developing strategies to obtain and extract additional information from inside the neutrophils. These are pilot studies and show great potential to get chemical information in a label free way and is a good complement to fluorescence, SEM and TEM. We then made an in-depth investigation on the mechanisms how nanoparticles interact with neutrophils, with special focus on processes triggering NETs formation. Using QDots as a model system we could show that the NETs release is strongly dependent on the uptake of the nanoparticles. We used fluorescence and TEM to investigate where the QDots uptake and to identify the pattern where they finally end up. We clearly observed them inside vesicles in the inner part of the cell and even within the NETs structure giving proof that the uptake of QDots play an important role of the NETs formation. In the last paper we expanded the study and exposed the cells to Iron Oxide NPs (FeNP) Here we developed a strategy how to alternate the magnetic field control the direction of the NETs. We could manipulate live NETs with a magnetic field and made observations that parts of the NETs are static and some clearly mobile, still with an internal memory to find its initial structure just after release. TEM studies revealed that, like the QDots, the FeNP end up inside the NETs. In conclusion in this thesis work, detailed processes are explored on neutrophils and their NETs formation with new unconventional methods and how neutrophils and nanoparticles interact with respect to NETs. 

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