Plasmonic Gap-Enhanced Raman Tag Nanorods for Imaging 3D Pancreatic Spheroids Using Surface-Enhanced Raman Spectroscopy (SERS) and Darkfield Microscopy.

Plasmonic gap-enhanced Raman tags (GERTs) are new emerging nanoprobes that based on their unique surface-enhanced Raman spectroscopy (SERS) signal can play a major role in complex imaging and detection of biological systems. GERTs are synthesized from a metal core nanostructure and layered with one or several additions of metal nanosized-layers, encasing a Raman active molecule. The advantages of GERTs are enhanced surface plasmon and electromagnetic resonance, but also inherent protection of the Raman active molecule from the environemental deterioration that could reduce their spectroscopic signatures over time. In this study, we choose in vitro 3D spheroids cultures to demonstrate these advantages. 3D spheroids mimic the in vivo tumor microenvironment better than 2D culture, with abundant extracellular matrix and hypoxia inducing variability of pH and enzymatic reactions. Here, we report the use of GERTs in large pancreatic 3D spheroids (>500 m in apparent diameter) for complex penetration visualization. Our combined imaging technique of enhanced darkfield microscopy and SERS was able to identify the presence and distribution of the GERTs within the 3D spheroid structure. The distribution of GERTs 2 hours after the nanorods incubation indicated accumulation, generally in the outermost layer of the spheroids but also, more randomly, in a non-uniform patterns in deep layers of the 3D spheroids. These observations bring into question the mechanism of uptake and flow of the nanoparticles in function of their incubation time while demonstrating the promising potentials of our approach. Additionnally, SERS signal was still detectable even after 24 hrs in incubation of GRETS interaction with 3D culture indicating a stability of the Raman signal.