Correlated light and electron microscopy (CLEM) has become essential in life sciences due to advancements in imaging resolution, sensitivity, and sample preservation. In nanotoxicology─specifically, studying the health effects of particulate matter exposure─CLEM can enable molecular-level structural as well as functional analysis of nanoparticle interactions with lung tissue, which is key for the understanding of modes of action. In our study, we implement an integrated high-resolution fluorescence lifetime imaging microscopy (FLIM) and hyperspectral fluorescence imaging (fHSI), scanning electron microscopy (SEM), ultrahigh resolution helium ion microscopy (HIM) and synchrotron micro X-ray fluorescence (SR μXRF), to characterize the nanobio interface and to better elucidate the modes of action of lung epithelial cells response to known inflammatory titanium dioxide nanotubes (TiO2 NTs). Morpho-functional assessment uncovered several mechanisms associated with extensive DNA, essential minerals, and iron accumulation, cellular surface immobilization, and the localized formation of fibrous structures, all confirming immunomodulatory responses. These findings advance our understanding of the early cellular processes leading to inflammation development after lung epithelium exposure to these high-aspect-ratio nanoparticles. Our high-resolution experimental approach, exploiting light, ion, and electron sources, provides a robust framework for future research into nanoparticle toxicity and its impact on human health.

Department of Electrochemical Materials J Heyrovský Institute of Physical Chemistry v v i Dolejškova 3 182 23 Prague 8 Czech Republic

Elettra Sincrotrone Trieste Strada Statale 14 km 163 5 in AREA Science Park Trieste 34149 Italy

Institute for Ion Beam Physics and Materials Research Helmholtz Zentrum Dresden Rossendorf e 5 Bautzner Landstrasse 400 01328 Dresden Germany

Jožef Stefan Institute Jamova cesta 39 1000 Ljubljana Slovenia

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High-Resolution Correlative Microscopy Approach for Nanobio Interface Studies of Nanoparticle-Induced Lung Epithelial Cell Damage