NANOBIOPHOTONICS APPROACHES TOWARDS ADVANCED BIOIMAGING

Due to its nature, imaging is dependent on data visualization. More specifically, when dealing with optical microscopy imaging, diffraction limited spatial resolution and the presence of out-of-focus contributions are two of the key elements for the generation of 4D (x,y,z,t) data sets oriented to study molecular processes at the nanoscale. Within the medical nanotechnology scenario, optical fluorescence techniques have a pivotal role thanks to their non invasive capability to address biological questions in three dimensions with a remarkable capability to distinguish fine details, tremendously improved up to the nanoscale level in the last decade. Within this scenario, this Thesis concerns the study and the development of advanced fluorescence optical methods for the improvement of the imaging capability and a better exploitation of its potentialities. Two approaches have been followed in fluorescence microscopy to gather information, namely: a direct diffraction-limited observation of the sample allowed by the optical architecture and the analysis of those fluorescence signals particularly sensitive to the environmental condition. Consequently, two strategies have been pursued to improve such capabilities. They are related to the design of novel optical arrangements utilizing light interferences pathways to improve the diffraction resolution limit and to the study of novel fluorescent molecules and/or fluorescent techniques which allows the investigations of subresolved molecular interactions. In particular, following a fluorescent probe approach, a nanostructured polyelectrolyte system has been designed to study the fluorescence quenching effect induced by specific quencher molecules on the fluorescence emission process. The realized system allows entrapping the fluorescent molecules and monitoring fluorescence signal variations to probe quencher metal ions at microrange concentrations, significantly higher with respect to the current fluorescent quenching based technologies. As well, following an optical approach, the interferences effect induced by structuring the illumination light with different masks have been studied, in order to improve some features of the imaging capability of the fluorescence microscope. In the Two-Photon Excitation (2PE) and in the Confocal Single Photon Laser Scanning Microscope, the insertion of a ring shaped mask in the illumination pathway is proposed to enhance the signal to noise ratio of the optical system at the high spatial frequencies. Since in these optical systems the transmission of the high spatial frequencies is particularly weak, such features allow to improve the overall practical capability of the confocal and 2PE system to distinguish fine details of the image. In the widefield microscope, the insertion of a periodic grid to structure the light has been investigated in order to confer a 3D optical sectioning capability comparable to that of the confocal microscope, with major advantages in terms of the efficient use of the light, simplicity of construction, speed of imaging acquisition, versatility, and low cost. The proposed scheme allows to quickly collect a pure sectioned image without any computational demodulation thanks to a novel optical architecture where both the illumination and the detection light is structured by a spinning grid.