[en] The microwaves electromagnetic properties, i.e. the complex permittivity, of single cells determine how this radiation is transmitted, reflected or absorbed by biological tissues. This information is important for the development microwave medical applications in diagnostics and therapy. Moreover, it is also crucial to assess the potential dangerous effects of the exposure to microwave radiations. Dielectric spectroscopy performed allowed to quantify the complex permittivity of tissues and whole single cells. However, there is a lack of information at the sub-cellular and intracellular level, due to the inherent limitations of the techniques, to resolve the dielectric response at the nanoscale. In recent years, Near-Field Scanning Microwave Microscopy (NF-SMM) has appeared as promising alternative to obtain images related to the dielectric response of samples, with high spatial resolution. In SMM, the local reﬂection of microwaves from the sample is measured by means of a sharp probe scanned in close proximity to the sample, i.e. within the near-ﬁeld region. The reflection can be related to the electrical impedance of the samples, and from this, the local complex permittivity can be retrieved. The near-field region ensures the good lateral resolution of the technique, far below the wavelength of the radiation used. SMM has been only scarcely applied to biological samples, and the few studies are limited to qualitative findings. This is due, among other reasons, to the complexity of the interpretation of the data, especially in case of tall irregular samples like cells, where the topography crosstalk effect dominates the signal acquired, thus masking the dielectric response. The objective of my Thesis was precisely to use this technique to quantify the local nanoscale dielectric response of a single cell at microwave frequency. My research focused primarily in the elaboration and implementation of the analysis methodologies suitable to obtain quantitative information from SMM measurements. I elaborated a methodology to disentangle and remove the topography crosstalk effect in the capacitance images acquired by SMM, which allows to extract new capacitance images only related to the intrinsic dielectric response of the sample, and therefore suitable for the quantification. I extracted the permittivity of the sample from the intrinsic capacitance images, by means of data analysis procedures which I adapted from the one available for low frequency measurements within the research group. Among these, the procedures to determine tip and sample geometries and to obtain the permittivity. The procedures were validated on reference samples. I first analysed heterogeneous inorganic thin film, exhibiting large height variations comparable to the ones of bacterial cells. I obtained intrinsic capacitance images at around 19 GHz in contact mode and show these can be directly related to the permittivity of the samples, without the need of theoretical models or the knowledge of the system geometry, and therefore represent maps of the microwave permittivity. I also show that in case of images acquired in intermittent contact mode the interpretation of the capacitance images in terms of the electric permittivity is much more complex. Finally, I obtained intrinsic images, at ~19 GHz, of a single E.coli bacterium, in dry and humid conditions, and, with the help of theoretical models, I extracted the local permittivity. These findings represent the first quantification of the of a single cell ever done at microwaves at the nanoscale, and thus show that SMM is sensitive to the cell constituent and the environment humidity. The results obtained prove that, despite the complexity of the data analysis, the microwave permittivity of biological samples can be quantified with nanoscale resolution, from SMM capacitance images.

[es] Actualmente, la mayoría de información sobre la interacción entre radiación de microondas y materia biológica ha sido proporcionada por estudios en tejidos o suspensiones celulares. Sin embargo, para superar la variabilidad de resultados asociada a la heterogeneidad de la materia biológica a escala macro y micrométrica, así como para evaluar los mecanismos fisiológicos que puedan originar efectos dañinos de la exposición a las radiaciones de microondas, es necesario estudiar la interacción a una escala mucho menor, es decir nanométrica, en células individuales, componentes subcelulares y macromoléculas. Recientemente la microscopia de rastreo con microondas en campo cercano (NF-SMM) ha aparecido como técnica prometedora para obtener imágenes con alta resolución espacial relacionadas con la respuesta dieléctrica de la muestra. Sin embargo, apenas ha sido aplicada a muestras biológicas, y de manera cualitativa. Para muestras de alturas irregulares como células, este hecho es debido, entre otras razones, a la presencia dominante de una contribución parásita (crosstalk topográfico), que a menudo puede enmascarar la respuesta dieléctrica local de la muestra. En esta tesis, el objetivo se ha enfocado en la elaboración y aplicación de metodologías de análisis para obtener información cuantitativa acerca de la permitividad local de una célula individual a las microondas, a partir de medidas de SMM. Antes de la aplicación a muestras biológicas de interés, las metodologías han sido aplicadas a muestras inorgánicas heterogéneas y tridimensionales, que al igual que las células individuales, presentan el problema del crosstalk topográfico. Finalmente, se han obtenido imágenes eléctricas, aproximadamente a ~ 19 GHz, de una bacteria E. coli, en condiciones ambientales secas y húmedas. Combinando las imágenes experimentales con modelos numéricos 3D y las herramientas de análisis desarrolladas, ha sido posible extraer la permitividad local de la célula. E. coli ha resultado ser una muestra no plana, y dieléctricamente homogénea. Se ha determinado un valor de permitividad dieléctrica alrededor de 4 en seco y de 20 en húmido, lo cual es consistente con los componentes biológicos de esta célula y la presencia de agua en el medio ambiente. Estos resultados representan la primera cuantificación de la permitividad local de una sola célula hecha en microondas.