Mapping Morphology-Dependent Stability of Gold Nanostars in Immune Cells Using Hyperspectral Imaging

Gold nanoparticles (AuNPs) are widely applied in nanomedicine, cellular and tissue biology, nanoscopy, photothermal therapy, and a range of diagnostic and clinical technologies. Among them, gold nanostars (AuNSs) have emerged as particularly promising due to their highly tunable optical and chemical properties. However, like other nanostructures, the stability of AuNSs remains a key challenge, especially within complex cellular microenvironments. Here, wide-field hyperspectral microscopy is evaluated for the real-time characterization of the morphology-dependent stability of AuNS formulations in immune-cell microenvironments. A computationally efficient image processing pipeline extracts statistical features from reflectance images, enabling the real-time analysis of hyperspectral data. UMAP-based visualization of spectral data revealed distinct, time- and formulation-dependent spectral shifts, with smaller seed volume formulations (larger overall diameter) for AuNSs exhibiting rapid destabilization and aggregation in THP-1 cells. In contrast, larger seed volume formulations (smaller overall diameter) for AuNS demonstrated enhanced colloidal stability and spectral uniformity. Compared to conventional ensemble measurements, hyperspectral reflectance measurements provided a rapid and resource-efficient approach that enabled macroscale imaging while retaining the spectral detail necessary to resolve AuNS transformations. Overall, the hyperspectral microscopy techniques presented here provide a label-free, high-throughput platform for evaluating AuNS stability and biocompatibility, with strong potential to guide the rational design of AuNSs for immunotherapeutic and diagnostic applications.