M24 - Multiplexed Spectral SERS Imaging of Functionalized Nanoparticles Comparing Transmission and Oblique Single Plane Illumination Microscopy

Advances in multiplex SERS imaging of cells are important for many biomedical challenges, such as disease biomarker detection and therapeutic monitoring. SERS imaging of biological cell samples has been demonstrated in the past; however, traditional point mapping and line imaging can be slow and can suffer from high background signals, making multiplexed detection difficult. The goal of our research is to develop a new spectral imaging method based oblique selected plane illumination microscope (oSPIM) to record wide-field SERS images and the spectra from nanoparticles simultaneously.  In this presentation, we demonstrate the ability to distinguish spectra of differently functionalized nanoparticles within a single sample and field of view. Gold nanostars (AuNS) functionalized with three different Raman reporters were synthesized including 4-mercaptobenzoic acid (MBA), 4-nitrobenzenethiol (NBT), and 2-mercaptobenzonitrile (MBN) due to their reliable spectral signatures. The functionalized nanoparticles were combined into one solution and suspended in a gelatin matrix above a glass coverslip. These types of samples were imaged on both the transmission and the oSPIM microscopes to simulate imaging in three-dimensional (3D) matrices and demonstrate proof of concept for cellular imaging. Using a rastered 660 nm diode laser and sequentially scanning throughout a sample generates the selected plane (a.k.a. lightsheet) illumination, from which the multiplex detection of distinct SERS signals is possible with minimal background. Comparison of results to the imaging and spectral capability of a transmission configuration suggests added benefits of lightsheet illumination for the reduction of out of focus light. The two microscope configurations, lightsheet and transmission, are spectrally-resolved widefield microscopes.  A transmission diffraction grating is located close to imaging sensor to simultaneously collect the zeroth order (n=0) and first order (n=1) collection, thus recording both the image and spectrum on a single sensor.  Spectral SERS imaging instrumentation suggests new bioimaging capabilities, such as tracking multiple SERS particles in cancer cells.