Imaging and visualization of cells and tissues are important when studying various biological phenomena. The ability to provide spatial information with molecular and chemical specificity may increase our insight and understanding of biological problems within life sciences. There is a need for well suited analytical imaging tools for addressing challenges that can increase our knowledge from the visualization on the cellular and subcellular level. In this thesis, we have focused on the use of surface analytical techniques based on the photoemission process. Synchrotron based surface analytical tools such as mirror electron microscopy, low energy electron microscopy, X-ray photoelectron spectroscopy, X-ray photoemission electron microscopy and near edge X-ray absorption fine structure spectroscopy were used to obtain highly resolved chemical information for both fundamental biological systems and technical innovations.

A combined photoemission electron microscopy and imaging X-ray photoelectron spectroscopy instrument have been used for visualization and characterization of neutrophils attached to silicon and gold surfaces. Neutrophils are white blood cells and a major part of our innate immune system. In the body they circulate and scavenge for possible threats, such as pathogens. The neutrophils possess three main defense mechanisms to tackle any possible threat in the body. One of these mechanisms is the release and formation of extracellular traps used for entrapping and capturing. We have visualized the extracellular trap formation in presence of nanoparticles and images of the neutrophils have been obtained with threshold mapping and work function contrast from energy-filtering operations together with element specific imaging and chemical maps. We demonstrated work function variation in imaging mode for the cellular morphology and the characteristic polymorphonuclear morphology of the nucleus. These results demonstrate the potential and extend the use of photoemission electron microscopy and imaging X-ray photoelectron spectroscopy as analytical tools for visualization of biological materials and processes on the cellular level.

The use of nanoparticles in recent years have significantly increased. Today, nanoparticles are being used in a wide range of applications, such as in electronics, energy, biology, and medicine. One hot topic in medicine is the development of contrast enhancement agents for magnetic resonance imaging. We report the development of two types of nanoparticles to be used as contrast enhancers for magnetic resonance imaging. The first type is water-dispersible and ultra-small Fe₃O₄ nanoparticles coated with polyacrylic acid. The Fe₃O₄ nanoparticles exhibit good magnetic properties, biocompatibility, excellent relaxivity properties and can be employed as a potential dual T₁ and T₂ weighted contrast agent. The second type is cerium oxide nanoparticles with the integration of gadolinium. Cerium oxide has unique redox properties due to the coexistence of Ce³⁺ and Ce⁴⁺ states making them suitable for scavenging reactive oxygen species. The integration of gadolinium makes these nanoparticles promising contrast agents with both therapeutic and diagnostic properties. We have designed a new technical innovative energy saving  process where a reduction in the annealing temperature for oxide removal is obtained, by the presence of europium doped gadolinium oxide nanoparticles in comparison to Eu³⁺ and Gd³⁺. A low coverage of nanoparticles and ions revealed a significant reduction in annealing temperature for the oxide removal. These results deliver a promising one step energy saving strategy of producing silicon-based contacts.

In summary, this thesis work demonstrates the power of element specific imaging and chemical mapping of bio-related surfaces as well as nanoparticle tracking in the sub-micro and nano region.

Avbildning av celler och vävnader är en viktig del i att studera biologiska processer. Förmågan att visualisera biologiska prover med molekylär och kemisk specificitet kan öka vår insikt och förståelse för de biologiska processerna. Idag finns det ett behov av att använda anpassade analytiska avbildningstekniker för att hantera dessa utmaningar som kan öka vår kunskap genom avbildning på den cellulära och molekylära nivån.

I detta avhandlingsarbete ligger fokus på avbildning av neutrofila granulocyter, en typ av vita blodkroppar med hjälp av ett fysikaliskt fenomen, den så kallade fotoelektriska effekten. Den fotoelektriska effekten kan förklaras genom att elektroner slås ut från ett material om man belyser det med ljus som har en tillräcklig hög frekvens. Dessa kallas då fotoemitterade elektroner. Vi har fokuserat på instrument som är baserad på den fotoelektriska effekten för visualisering och karakterisering av neutrofiler. Neutrofiler är den vanligaste celltypen av vita blodkroppar och fyller en mycket viktig funktion i det medfödda immunförsvaret. Dessa celler cirkulerar i blodet och är bland de första vid en akut inflammation. Neutrofilernas främsta uppgifter är att ta hand om och oskadliggöra främmande patogener. De använder sig av olika typer av försvarsstrategier och en av dessa består av att neutrofilen bildar och kastar ut ett nätverk som består av dess egna DNA.

Vi har specifikt använt oss av ett instrument för avbildning av neutrofiler som kombinerar teknikerna från både fotoemissionelektronmikroskopi och avbildande röntgenfotoelektronspetroskopi. Dessa instrument erbjuder ytanalyser och skapar en kraftigt förstorad bild genom de fotoemitterade elektronerna. Utöver den förstorade bilden ges spatial information om olika atomslag och den elektroniska strukturen hos de ingående molekylära komponenterna.

Dessa resultat belyser potentialen och utökar användningen av fotoemissionselektronmikroskopi och avbildande röntgenfotoelektronspektroskopi som analysverktyg för visualisering av biologiska material och processer på cellnivå.

Nanotekniken och användningen av nanopartiklar har under de senaste åren ökat markant. Nanopartiklar är små partiklar med en diameter på mindre än några miljarddels meter. Idag används nanopartiklar i en uppsjö av olika tillämpningar, till exempel inom elektronik, energi, biologi och medicin. En utmaning idag är att tillverka och utveckla kontrastförstärkande medel baserade på nanopartiklar för användning inom bildgivande magnetresonanstomografi, en teknik för avbildning av mjuk vävnad. I denna avhandling har vi utvecklat två typer av kontrasförstärkande medel där den ena består av extremt små nanopartiklar av järnoxid som genom dess storlek kan användas som både ett positivt och negativt kontrastmedel och där den andra består av nanopartiklar som baseras på ceriumoxid med integrerat gadolinium. Dessa nanopartiklar har antioxidativa egenskaper på grund av samexistens av två oxidationstillstånd. Detta möjliggör ett kontrastmedel med både terapeutiska och diagnostiska egenskaper.

En annan tillämpning av nanopartiklar som vi har undersökt är reducering av annealingtemperaturen av kiseloxid med hjälp av närvaron av europium-dopade gadoliniumoxid-nanopartiklar. En låg täckning av nanopartiklar och joner avslöjade en signifikant minskning av annealingtemperaturen för oxidavlägsning. Synkrotronljusbaserade tekniker har använts för att karakterisera ytan med högupplöst kemisk information. Under annealingsprocessen inträffade en fragmentering av nanopartiklarna där gadolinium migrerade till kiseloxidrika områden, medan europium migrerade till rena kiselområden. Dessa resultat visar ett lovande energisparande sätt att producera kiselbaserade kontakter.

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.