Exploring how tiny particles affect our lungs

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.

Figure 1: Novel multimodal microspectroscopy-based correlative microscopy with the typical imaging ranges for each technique enables a comprehensive investigation of the studied nanobio interface across a broad spatial scale, from tens of microns down to the nanometer level. Adapted from R. Podlipec et al., 10.1021/acsnano.4c17838 (2025), published in ACS Nano, licensed under CC BY 4.0).

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).

The data indicate that lung cells interact with these nanoparticles in complex ways. The particles tend to form clusters on the cell surface (Fig. 2a) and bind to biological materials such as DNA. These clusters or composites may play a key role in triggering immune responses—both inflammatory and anti-inflammatory. One of the early responses we observed was the formation of fibrin-like fibrous networks over the particles (Fig. 2b), plausibly marking the beginning of an acute reaction by the cells. We also discovered that the particles can cause the accumulation of DNA, essential minerals, and iron (Fe) in certain areas of the cell surface (Fig. 2c), further supporting the idea that these particles influence immune responses.

Figure 2: Multimodal CLEM-μXRF revealing physicochemical properties of the nanobio interface formed on the surface of lung epithelial cells. (a) Multichannel 3D CLSM on live cells showing size, shape and topography of the nanobio composite. (b) High-resolution SEM on top of the CLSM showing the local formation of plausibly inflammatory fibrin-like fibrous structures over TiO₂-bio composites. (c) Correlated SR μXRF, FLIM and SEM demostrating causality between the local accumulation of Fe and local molecular (physical) changes in the surrounding biological environment, as measured by FLIM. Adapted from R. Podlipec et al., 10.1021/acsnano.4c17838 (2025), published in ACS Nano, licensed under CC BY 4.0).

Although using so many different imaging techniques and methods — some of which required live cells and others fixed (preserved) samples—posed technical challenges, we successfully combined the data from all approaches. This provided a powerful and detailed picture of how these particles interact with lung tissue. 

In conclusion, the correlative microscopy approach, using light-, ion-, and electron-based techniques at high resolution, offers a valuable tool for future research into how nanoparticles affect human health and how we might better protect ourselves from their potential risks.

This research was conducted by the following research team:

Rok Podlipec^1,, Luka Pirker^1,3, Ana Krišelj¹, Gregor Hlawacek², Alessandra Gianoncelli⁴ and Primož Pelicon¹

¹ Jožef Stefan Institute, Ljubljana, Slovenia.
² Helmholtz-Zentrum Dresden-Rossendorf e.V., Ion Beam Center, Dresden, Germany.
³ J. Heyrovský Institute of Physical Chemistry, Department of Electrochemical Materials, Prague, Czech Republic.
⁴ Elettra, Sincrotrone Trieste S.C.p.A., Trieste, Italy.

Contact person email:

Local contact person email: alessandra.gianoncelli@elettra.eu