Advanced super-resolution microscopy for quantifying cytoskeletal responses in FLASH radiotherapy

This thesis develops quantitative methodologies employing fluorescence and super-resolution imaging to study cytoskeletal remodeling induced by radiation in healthy and cancer cells, contributing to research on FLASH radiotherapy — an innovative therapeutic approach capable of achieving tumor control comparable to conventional radiotherapy while significantly mitigating radiation-induced toxicity in healthy tissues. While cytoskeletal networks are critical for cellular function, they are understudied in radiotherapy compared to DNA damage and free radicals. Existing protocols analyze specific cytoskeletal features, but a comprehensive tool for characterizing radiation-induced cytoskeletal responses is lacking. To address this gap, this thesis introduces imaging and analysis pipelines to investigate multiple cytoskeletal components and mechanisms driving epithelial-to-mesenchymal transition. Single Molecule Localization Microscopy was used to visualize nanometric structures such as microtubules, while advanced epifluorescence microscopy imaged vimentin and actin filaments. A computational pipeline was then developed to quantify the levels of cytoskeletal proteins, their spatial distributions, anisotropy, nanoscale organization, and the amounts of actin protrusions. The developed pipelines were applied to study healthy and cancer lung cells, yielding results across varying doses and irradiation modalities. Furthermore, an AI-assisted dual-color super-resolution method was developed to visualize dynein motors and microtubules at the nanoscale, with the aim of applying it to future quantitative studies of molecular motors in radiotherapy.