Nanoscale electrical characterization of biological matter at microwave frequencies

[eng] 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 re? ection 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-? eld 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. [spa] Actualmente, la mayoria de informacion sobre la interaccion entre radiacion de microondas y materia biologica 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 biologica a escala macro y micrometrica, asi como para evaluar los mecanismos fisiologicos que puedan originar efectos daninos de la exposicion a las radiaciones de microondas, es necesario estudiar la interaccion a una escala mucho menor, es decir nanometrica, en celulas individuales, componentes subcelulares y macromoleculas. Recientemente la microscopia de rastreo con microondas en campo cercano (NF-SMM) ha aparecido como tecnica prometedora para obtener imagenes con alta resolucion espacial relacionadas con la respuesta dielectrica de la muestra. Sin embargo, apenas ha sido aplicada a muestras biologicas, y de manera cualitativa. Para muestras de alturas irregulares como celulas, este hecho es debido, entre otras razones, a la presencia dominante de una contribucion parasita (crosstalk topografico), que a menudo puede enmascarar la respuesta dielectrica local de la muestra. En esta tesis, el objetivo se ha enfocado en la elaboracion y aplicacion de metodologias de analisis para obtener informacion cuantitativa acerca de la permitividad local de una celula individual a las microondas, a partir de medidas de SMM. Antes de la aplicacion a muestras biologicas de interes, las metodologias han sido aplicadas a muestras inorganicas heterogeneas y tridimensionales, que al igual que las celulas individuales, presentan el problema del crosstalk topografico. Finalmente, se han obtenido imagenes electricas, aproximadamente a ~ 19 GHz, de una bacteria E. coli, en condiciones ambientales secas y humedas. Combinando las imagenes experimentales con modelos numericos 3D y las herramientas de analisis desarrolladas, ha sido posible extraer la permitividad local de la celula. E. coli ha resultado ser una muestra no plana, y dielectricamente homogenea. Se ha determinado un valor de permitividad dielectrica alrededor de 4 en seco y de 20 en humido, lo cual es consistente con los componentes biologicos de esta celula y la presencia de agua en el medio ambiente. Estos resultados representan la primera cuantificacion de la permitividad local de una sola celula hecha en microondas.