Feasibility of label-free multiphoton luminescence imaging to track the fate of inhaled gold nanoparticles in

Significance: Understanding the biological journey of inhaled gold nanomaterials is critical for nanotoxicology and therapeutic delivery. Conventional tracking methods often require fluorescent labeling, which can alter nanoparticle (NP) surface chemistry and lead to "false-positive" signals if the label dissociates. Aim: In this feasibility study, we utilize multiphoton luminescence (MPL) imaging to track the long-term biodistribution of 50 nm AuNPs following inhalation exposure at 1 week and 1 month. Approach: Mice were administered AuNPs via intranasal inhalation in four experimental groups: low (0.1 nM), medium (1 nM), high (10 nM) doses for 1 week, and a medium dose (1 nM) for 1 month. Organs were harvested and analyzed using ICP-MS for total gold content and MPL imaging for label-free visualization of AuNP deposition. Results: AuNPs remained localized within the lungs with no evidence of systemic translocation to the liver, spleen, brain, or kidneys at either time point. MPL imaging revealed that lung concentration followed a linear scaling model at one week. MPL imaging also revealed the long-term identification and localization of AuNPs within the lung at 1 month postinhalation. Conclusions: MPL imaging is superior to brightfield microscopy for NP detection, enabling the visualization of dose-dependent aggregation and deep-tissue mapping. These findings demonstrate the feasibility of MPL imaging as a label-free complementary imaging approach for pulmonary nanoparticle biodistribution studies.