Multiplexed Molecular Imaging of Fresh Tissue Surfaces Enabled by Convection‐Enhanced Topical Staining with SERS‐Coded Nanoparticles

Abstract

There is a need for intraoperative imaging technologies to guide breast-conserving surgeries and to reduce the high rates of re-excision for patients in which residual tumor is found at the surgical margins during post-operative pathology analyses. Feasibility studies have shown that utilizing topically applied surface-enhanced Raman scattering (SERS) nanoparticles (NPs), in conjunction with the ratiometric imaging of targeted vs. untargeted NPs, enables the rapid visualization of multiple cell-surface biomarkers of cancer that are over-expressed at the surfaces of freshly excised breast tissues. In order to reliably and rapidly perform multiplexed Raman-encoded molecular imaging (REMI) of large numbers of biomarkers (with 5 or more NP flavors), an enhanced staining method has been developed in which tissue surfaces are cyclically dipped into a NP-staining solution and subjected to high-frequency mechanical vibration. This dipping and mechanical vibration (DMV) method promotes the convection of the SERS NPs at fresh tissues surfaces, which accelerates their binding to their respective biomarker targets. By utilizing a custom-developed device for automated DMV staining, we demonstrate the ability to simultaneously image 4 cell-surface biomarkers of cancer at the surfaces of fresh human breast tissues with a mixture of 5 flavors of SERS NPs (4 targeted and 1 untargeted control) topically applied for 5 min and imaged at a spatial resolution of 0.5 mm and a raster-scanned imaging rate of >5 cm2/min.

Raman-Encoded Molecular Imaging with Topically Applied SERS Nanoparticles for Intraoperative Guidance of Lumpectomy

“…S1A and B)(28,30,31,33,41,42) and, more importantly, provides a quantitative measure of biomarker expression levels that agrees with both flow cytometry and gold-standard immunohistochemistry (Supplementary Fig. S2A–D)(28,30,32). …”

“…By functionalizing various flavors of SERS NPs with different targeting molecules (e.g. antibodies (28), affibodies (29), etc. ), the NPs can be multiplexed to simultaneously target and image a large panel of protein biomarkers.…”

“…We have recently demonstrated that a Raman-encoded molecular imaging (REMI) technique, which utilizes the topical application of multiplexed SERS NPs, enables the rapid visualization of multiple cell-surface biomarkers at the surfaces of fresh tissues (28,30–32). In our REMI approach, toxicity and sterility concerns are circumvented by staining and imaging fresh surgical specimens ex vivo , with the potential to allow for expedited regulatory approval and rapid clinical translation.…”

Surface-Enhanced Raman Scattering Nanoparticles for Multiplexed Imaging of Bladder Cancer Tissue Permeability and Molecular Phenotype

“…24,28 The active-to-passive normalization technique exhibited a moderate classification accuracy that was hampered by experimental noise in the Raman signal (Figure 7). To overcome limitations imposed by noise, we designed an alternative approach where the signal from actively targeted nanoparticles was normalized to the sum of all three nanoparticles channels.…”

Rinsing paired-agent model (RPAM) to quantify cell-surface receptor concentrations in topical staining applications of thick tissues

“…, with fluorescence (Davis et al , 2013) or surface-enhanced Raman scattering (SERS) agents (W Wang et al , 2016)) without the significant sample processing required in conventional immunohistochemistry, tissue microarrays, or mass spectrometry of non-vital tissues (Dabbs, 2013; Kononen et al , 1998; Lockhart and Winzeler, 2000; Stoeckli et al , 2001; Cukierman et al , 2001). Theoretically, such methods could be carried out with various imaging systems including clinical imaging systems (Keating et al , 2016), confocal fluorescence microscopy (Schiffhauer et al , 2009; Dobbs et al , 2016), spectrally encoded confocal microscopy (Brachtel et al , 2016), multi-photon microscopy (Zipfel et al , 2003), super-resolution microscopy (Huang et al , 2008), wide-area optical-sectioning structured illumination microscopy (SIM) (Fu et al , 2013), and microscopy with UV surface excitation (Levenson et al , 2016).…”