ILC report on the identification, localization and quantification of inorganic nanoparticles in tissue phantom samples

This report presents the results of an internal interlaboratory comparison (ILC) conducted within the MetrINo project as deliverable D8 to cross-validate orthogonal analytical techniques for the detection, localization, and quantification of inorganic nanoparticles (NPs) in biological matrices. The primary goal was to correlate complementary methodologies and define measurement uncertainties across a gradient of biological complexity.

The ILC utilized four distinct sample categories: homogeneous epoxy phantoms for instrument calibration; 3D engineered tumor models using SISmuc scaffolds; HfO$_2$-spiked mouse liver tissue; and A549 lung cancer cells (analyzed as both 2D deposits and suspensions).

The analytical framework integrated several complementary modalities: 

• Imaging and Localization: Two-Photon Excitation Fluorescence (TPEF) microscopy was used for deep-tissue 3D imaging, while Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) and SEM-EDX provided high-resolution surface chemical and elemental mapping.
• Quantification: Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) provided spatially resolved elemental quantification. For cellular mass analysis, the report compared bulk ICP-MS/OES with Single-Cell ICP-MS (SC-ICP-MS).

Key results include the development of a custom Python-based registration pipeline using a KDTree-based centroid matching algorithm, which successfully correlated TPEF optical intensities with LA-ICP-MS and ToF-SIMS data. This allowed for the identification of optimal TPEF instrument settings to translate fluorescence counts into absolute nanoparticle concentrations. In cellular studies, LA-ICP-MS and bulk ICP-MS demonstrated strong agreement in quantifying HfO$_2$ uptake, whereas SC-ICP-MS faced significant challenges due to low transport efficiency and cell disruption during the injection process.

By validating these orthogonal approaches, the report establishes the technical requirements and metrological foundation necessary for a future external ILC within the VAMAS TWA 40 framework.