• Biophysics
• Cytoskeleton
• Fluorescence Microscopy
• Radiotherapy
• Super-resolution Microscopy

In these recent years, fluorescence microscopy techniques rapidly evolved and became powerful tools to study biological structures and bio-molecular arrangements at the nanoscale. This thesis has developed and optimized imaging protocols to fully characterize distinct features of cellular cytoskeleton networks1. Intensity-based fluorescence imaging allows for cytoskeletal proteins expression estimation, while machine-learning based super-resolution imaging methods performs quantitative studies of the filaments’ orientation. A multilevel approach based on wide-field microscopy, highly inclined and laminated optical sheet (HILO) microscopy and AI-assisted super-resolution microscopy will be used to study both cytoskeletal intermediate filaments (i.e. via vimentin) and microtubules networks (i.e. by means of α-Tubulin protein).

The developed method helped elucidating the mechanism behind the sparing effect of FLASH-RT, a new and innovative radiotherapy technique. FLASH-RT, different from conventional radiotherapy (CONV-RT), delivers a single ultra-high dose at a high dose rates to achieve similar tumor growth control efficacy to CONV-RT but significantly reducing the detrimental injury associated to it. The differences between FLASH-RT and CONV-RT are evident, but the full understanding of the effects requires further research since its biophysical mechanism remains largely unclear. Hence, we used our method to image cytoskeletal proteins in lung cancer and healthy cell lines, irradiated in both FLASH-RT and CONV-RT modality, shedding new light on the 'FLASH effect'.