Multimodal optical imaging strategy to evaluate the microscopic biodistribution of SERS gold nanoparticles across histology sections.

Significance: Surface-enhanced Raman scattering (SERS) gold nanoparticles (AuNPs) are widely used for molecular imaging and diagnostics, but their non-biodegradable nature necessitates accurate, spatially resolved methods to assess long-term biodistribution. Existing approaches often lack sensitivity and spatial resolution or require destructive sample preparation. Aim: We evaluate multiphoton luminescence (MPL) microscopy as a label-free approach for detecting SERS AuNPs in biological tissues while using Raman imaging for spectroscopic validation of nanoparticle (NP) localization. Approach: SERS AuNPs were intravenously administered in mice at multiple doses and analyzed at 1- and 8-week time points using MPL and Raman microscopy. Quantitative analyses were used to compare NP signals across organs, doses, and time points. Results: MPL and Raman imaging both detected SERS AuNP-associated signals across all doses, with strong spatial correlation observed among modalities. Dose-dependent increases in signal were observed for both modalities, with normalized positive pixel area fraction in liver increasing from 0.02 at 0.1 nM to 0.99 at 5 nM. Whole-organ mapping revealed preferential accumulation in the liver and spleen, while longitudinal analysis demonstrated increased NP aggregation at later time points. Conclusions: MPL enables sensitive, label-free detection of SERS AuNPs and, when combined with Raman imaging, provides a non-destructive framework for spatially resolved biodistribution analysis.