Novel correlative microscopy approach for nano-bio interface studies of ultrafine particle-induced lung epithelial cell damage

Correlative light and electron microscopy (CLEM) has attracted widespread interest in the life sciences community, particularly in light of recent developments in imaging resolution and sensitivity, and sample preservation. One of the highly relevant scientific areas that can be addressed by CLEM is nanotoxicology, the study of the environmental and, in particular, health effects of the ubiquitous particulate matter in polluted air. To assess safety and predict outcomes, particularly those induced by highly penetrating ultrafine particles (UFPs), it is invaluable to understand the mode of action at the molecular level, from the moment the particles interact with lung tissue. In our study we address these challenges by combining novel multimodal high-resolution live and fixed cell microscopy, from fluorescence lifetime imaging microscopy (FLIM), hyperspectral fluorescence imaging (fHSI), scanning electron microscopy (SEM), ultra-high resolution helium ion microscopy (HIM) to synchrotron micro X-ray fluorescence (SR µXRF). These provided a comprehensive insight into the structural and functional properties of the nano-bio interface formed on the surface of lung epithelial cells exposed to titanium dioxide nanotubes (TiO ₂ NTs) and for the first time, we report several important initial cellular responses. Besides the characterization of extensive biomolecule binding to TiO₂, including DNA and its accumulation-related plausible implications for apoptosis and inflammation, we show acute changes due to fibrin-like fibrous network formation over aggregated nano-bio composites and accumulated iron-induced changes in the molecular environment at the nano-bio interface. The applied CLEM approach demonstrates high applicability for studying the initial cellular responses to nanoparticles, which can play a crucial role in the body’s immune response and the development of inflammation. The methodology provides a robust framework for future research into nanoparticle toxicity and its impact on human health.