Dark-field hyperspectral imaging of single plasmonic gold nanorods and their scattering characteristics in complex biological environments

Adipose tissue derived stem cells (ASCs) has applications in soft tissue replacement-based tissue engineering. ASCs can potentially reduce many of the disadvantages of autologous fat transplantation such as donor-site morbidity and immune system rejection. Although, ASCs hold clinical relevance as a potential cell therapy candidate, widespread use of them is hampered due to inadequate data on the fate of stem cells after transplant. Hence a method to facilitate long term tracking of the cells will enable better understanding of stem cell fate in stem cell-based therapeutics. Here, we employ biocompatible surface functionalized nanorods for tracking the adipogenesis and osteogenesis differentiation of ASCs. Anisotropic plasmonic nanostructures based on silver (Ag) and gold (Au) have received much attention owing to their tunable size and shape dependent localized surface plasmon resonance (LSPR) with multiple applications such as biological contrast agents, photothermal conversion, plasmon-enhanced spectroscopies, optical sensors and in catalysis. Hyperspectral microscopy combining both nanoscale imaging and spectral characteristics from plasmonic nanostructures provides a powerful tool for their identification and quantitative spectral analysis of plasmonic nanostructures with unprecedented level of details. Here, we present the analysis of single particle spectroscopy of gold nanorods and their orientation dependent scattering properties using hyperspectral microscopy and validated with correlated high-resolution electron microscopy. Fairly monodisperse gold nanorods with bright longitudinal SPR centered at about 663 nm were synthesized using bromide-free surfactant mixture consisting of cetyltrimethylammonium chloride and sodium oleate. The nanorods were successfully characterized by UV-Visible spectroscopy, DLS, XPS, and TEM results. Dark-field hyperspectral and second harmonic generation (SHG) microscopy were performed on individual gold nanorods and their optical scattering spectra were analyzed for imaging orientation of single nanorods. The initial results revealed scattering spectra from individual gold nanorods displayed measurable spectral-shifts from their collective LSPR spectrum from bulk measurements performed using UV-Visible spectroscopy. The analysis and utility of gold nanorods for labeling stem cells and the orientation dependent spectral features of nanorods inside the cells will be characterized and discussed in detail. The cell viability, differentiation capacity, gene expression, potential cytotoxicity due to nanorods such as inflammatory molecule and reactive oxygen species production, adipogenic and osteogenic potential will be evaluated using histochemical staining and quantitative polymerase chain reaction (qPCR). The study has implications towards tracking individual nanorods in complex biological systems and beyond.