Very small particles — either naturally occurring in the air or engineered for industrial purposes — can penetrate deep into our lungs because they are smaller than 100 nanometers (about 1,000 times thinner than a human hair). These particles can cause serious long-term health problems, such as inflammation, and tissue damage, which can lead to fibrosis or even cancer.
To assess the health risks of these nanoparticles and predict how they might affect us, it is essential to understand exactly how they interact with lung cells at the molecular level — from the moment they come into contact with lung tissue. Such a detailed investigation requires sophisticated imaging techniques that can reveal both the structure and function of these tiny interactions. One powerful method is Correlated Light and Electron Microscopy (CLEM), which combines different types of microscopes to provide a more complete picture. Thanks to recent advances in resolution and sensitivity, CLEM has become an important tool in biological research.
In our study, we developed a new, expanded CLEM approach that combines several advanced imaging techniques to better understand how lung cells respond to a specific type of nanoparticle: titanium dioxide nanotubes (TiO₂ NTs), which are known to cause inflammation and are considered potentially carcinogenic. Our approach integrates a wide range of complementary tools, providing a morpho-functional assessment of the studied interface (Fig. 1):
- Confocal Laser-Scanning Microscopy (CLSM), together with Fluorescence Lifetime Imaging Microscopy (FLIM) and Hyperspectral Fluorescence Imaging (fHSI) to study live cells at the organelle and nanoscopic scales.
- Scanning Electron Microscopy (SEM) and Helium Ion Microscopy (HIM) for extremely detailed surface imaging.
- Synchrotron-based X-ray Fluorescence (SR μXRF) combined with Scanning Trasnmission X-ray Microscopy (STXM) for analyzing the chemical elements in and around the cells at submicrometric length scales.
Among these, SR μXRF has crucial importance, as it provides chemical sensitivity. Together, these methods allowed us to study interactions across many scales — from whole cells down to individual molecules.
Scanning transmission X-ray Microscopy (STXM) combined with low energy micro-X-ray Fluorescence (LE-μXRF) were carried out at the TwinMic beamline of Elettra. These measurements were complemented by Correlative light,electron and ion microscopy, namely fluorescence lifetime imaging microscopy (FLIM), hyperspectral fluorescence imaging (fHSI), scanning electron microscopy (SEM) and ultra-high resolution helium ion microscopy (HIM).
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