Expanding the Multiplexing Capabilities of Raman Imaging to Reveal Highly Specific Molecular Expression and Enable Spatial Profiling

Abstract

Profiling the heterogeneous landscape of cell types and biomolecules is rapidly being adopted to address current imperative research questions. Precision medicine seeks advancements in molecular spatial profiling techniques with highly multiplexed imaging capabilities and subcellular resolution, which remains an extremely complex task. Surface-enhanced Raman spectroscopy (SERS) imaging offers promise through the utilization of nanoparticle-based contrast agents that exhibit narrow spectral features and molecular specificity. The current renaissance of gold nanoparticle technology makes Raman scattering intensities competitive with traditional fluorescence methods while offering the added benefit of unsurpassed multiplexing capabilities. Here, we present an expanded library of individually distinct SERS nanoparticles to arm researchers and clinicians. Our nanoparticles consist of a ∼60 nm gold core, a Raman reporter molecule, and a final inert silica coating. Using density functional theory, we have selected Raman reporters that meet the key criterion of high spectral uniqueness to facilitate unmixing of up to 26 components in a single imaging pixel in vitro and in vivo. We also demonstrated the utility of our SERS nanoparticles for targeting cultured cells and profiling cancerous human tissue sections for highly multiplexed optical imaging. This study showcases the far-reaching capabilities of SERS-based Raman imaging in molecular profiling to improve personalized medicine and overcome the major challenges of functional and structural diversity in proteomic imaging.

Highly Excretable Gold Supraclusters for Translatable In Vivo Raman Imaging of Tumors

“…Raman spectroscopy identifies the distinctive spectral fingerprint of molecules, thus providing high specificity for biomedical imaging. − There has been significant advancements for in vivo preclinical Raman imaging in various applications, , from intraoperative imaging of microscopic tumors − to multiplexed imaging in living subjects. − Because the Raman scattering from molecules is generally weak, gold nanoparticles are employed to amplify the Raman signals through the localized surface plasmon-generated electromagnetic field, which is known as surface-enhanced Raman scattering (SERS). − Since the degree of the field enhancement is proportional to the size of metallic nanoparticles, large gold nanoparticles over 50 nm in diameter have been utilized to achieve sufficient sensitivity for in vivo imaging . The large gold nanoparticles exhibit minimal or negligible acute toxicity after systematic administration .…”

Multiplexed molecular imaging with surface enhanced resonance Raman scattering nanoprobes reveals immunotherapy response in mice via multichannel image segmentation

“…Multiplexed imaging of 5 untargeted nanoparticles at different ratios simultaneously in vivo has been reported, 23 as well as the spatial encoding of 26 distinct nanoparticles with targeting shown for 5 targets in vitro. 24 Here, we report a general framework for employing the technique for imaging the tumor microenvironment. Specifically, we present how to select Raman reporter molecules for SERS nanoprobe synthesis, a quantitative way of imaging formation by color-coding the multichannel pixel-wise spectra, an example application of 8-plexed imaging in a mouse model of cancer immunotherapy, and an algorithmic approach for parsing and classifying the multiplexed images based on machine learning (ML).…”

“…Multiplexed imaging of 5 untargeted nanoparticles at different ratios simultaneously in vivo has been reported, 23 as well as the spatial encoding of 26 distinct nanoparticles with targeting shown for 5 targets in vitro . 24…”

Design, Rationalization, and Automation of a Catalytic Sensing Mechanism for Homogeneous SERS Biosensors

“…SERS sensors have an optical readout, as currently used in the common colorimetric and fluorescence methods, but they permit highly multiplexed analysis due to the vibrational nature of the SERS phenomenon. Recent advances have shown simultaneous detection of 26 SERS reporters, in contrast to the three to four multiplexable dyes in fluorescence-based assays . In addition, SERS spectra are less prone to spectral background interference due to the sharp nature of SERS peaks compared to the broad background signal from autofluorescence and/or stray background light.…”

“…Recent advances have shown simultaneous detection of 26 SERS reporters, in contrast to the three to four multiplexable dyes in fluorescence-based assays. 4 In addition, SERS spectra are less prone to spectral background interference due to the sharp nature of SERS peaks compared to the broad background signal from autofluorescence and/or stray background light. The latter issue is particularly significant in POC applications where measurements are performed outside of ideal lab settings.…”