Surgical Guidance via Multiplexed Molecular Imaging of Fresh Tissues Labeled with SERS-Coded Nanoparticles

Abstract

The imaging of dysregulated cell-surface receptors (or biomarkers) is a potential means of identifying the presence of cancer with high sensitivity and specificity. However, due to heterogeneities in the expression of protein biomarkers in tumors, molecular imaging technologies should ideally be capable of visualizing a multiplexed panel of cancer biomarkers. Recently, surface-enhanced Raman-scattering (SERS) nanoparticles (NPs) have attracted wide interest due to their potential for sensitive and multiplexed biomarker detection. In this review, we focus on the most recent advances in tumor imaging using SERS-coded NPs. A brief introduction of the structure and optical properties of SERS NPs is provided, followed by a detailed discussion of key imaging issues such as the administration of NPs in tissue (topical versus systemic), the optical configuration and imaging approach of Raman imaging systems, spectral demultiplexing methods for quantifying NP concentrations, and the disambiguation of specific vs. nonspecific sources of contrast through ratiometric imaging of targeted and untargeted (control) NP pairs. Finally, future challenges and directions are briefly outlined.

Keywords: Raman spectroscopy; biomarkers; biomedical optical imaging; cancer detection; fiberoptic probes; molecular imaging; nanomedicine; tumors.

Fig. 1

SERS NPs and their application for tumor detection. (a) Typical structure of SERS NPs. (b) Functionalization of SERS NPs with targeting molecules. Schematics showing (c) topical administration, (d) systemic administration, (e) imaging and (f) demultiplexing of multiplexed SERS NPs.

Fig. 2

Penetration of SERS NPs on tissue and cells. (a) Bright-field and fluorescence images showing that topically applied SERS NPs (conjugated to a fluorophore cyto-647 in this case) are localized to the tissue surface of a piece of mouse muscle that was topically stained with EGFR-targeted SERS NPs (300 pM, 10 min), rinsed in PBS (20 s) and embedded in O.C.T. (optimal cutting temperature) compound. The image shows a frozen section (10-μm thick) that was imaged with a fluorescence microscope. (b) Bright-field and fluorescence confocal microscopy images showing the distribution of EGFR-targeted NPs on A431 cells. In this thin confocal image section (~ 5 micron section thickness), the fluorescence signal is primarily localized at the periphery of the cells, indicating that the NPs are at the cell surface. The scale bars represent 50 μm..

Fig. 3

Nonspecific and specific binding of SERS NPs topically applied on cultured cells and tissues. The bright-field images (first row) and fluorescence images (second row) of NP-stained cells, and the Raman images of NP-stained tissues (third row) are shown. The green and red colormaps indicate the fluorescence intensity and Raman intensity from the SERS NPs, respectively. The bottom images show the measured concentration ratio of EGFR-NPs vs. isotype-NPs, in which the nonspecific accumulation of the NPs is normalized away and the EGFR expression is clearly revealed. The unlabeled scale bars represent 20 μm.

Fig. 4

A common optical configuration for the imaging of SERS NPs in tissues.

Fig. 5

Schematic of imaging approaches. The labels c₁ - c_n represent the wavelength channels selected for wide-field imaging.

Fig. 6

Imaging of human breast tissues stained with a 2-flavor NP mixture (HER2-NPs and isotype-NPs, 150 pM/flavor). (a) Background spectra collected at different locations from the tissue specimen marked in (e). (b) The first three principal components and average background spectrum of the 162 background spectra collected from the specimen. (c) Example of fitted spectra and corresponding residuals when using Method 1 and Method 2. (d) The fitting error for all 2000 spectra when using the two fitting algorithms and two assessment metrics. (e) Photograph of a freshly resected breast tissue specimen. (f) Image of the concentration of HER2-NPs. (g) Image of the concentration ratio of HER2-NPs vs. isotype-NPs. (h) IHC staining with an anti-HER2 mAb. The scale bars represent 2 mm.

Fig. 7

Limit-of-detection (linearity) test by imaging four multiplexed SERS NPs. Four NP flavors were mixed in 1:1:1:1 ratio and diluted from 200 pM (per flavor) down to 1 pM. A 0.5-μL drop from each sample was imaged, and 3 spectra were acquired and demultiplexed to analyze the weight of each NP flavor. The concentrations were calculated based on the measured weights of stock NP mixture with known concentrations. (a) The reference spectra of the four NP flavors. (b) Raw spectra from NP mixtures at a concentration of 10 pM, 5 pM, 1 pM and 0 pM (water background). (c) Measured NP concentrations. (d) Concentration ratios. The measured concentrations show good linearity from 1 to 200 pM when 4 flavors of NPs are multiplexed, with larger errors in terms of concentration (~20%) and concentration ratio (~30%) appearing at 1 pM. (e–g) Imaging of 4-flavor NP mixtures. (e) A 2-μL drop from each dilution, and a drop from each undiluted NP flavor, were imaged to measure (f) the NP concentrations and (g) concentration ratios.

Fig. 8

In vivo endoscopic molecular imaging performed with multiplexed SERS NPs delivered via oral gavage. (a) Photograph of a surgically exposed rat esophagus implanted with three tumor xenografts. (d) Images showing the concentration ratio of EGFR-NPs vs. isotype-NPs and HER2-NPs vs. isotype-NPs. (c) Plots showing the correlation between the image-derived ratios from various tissue types (normal esophagus and three tumors) and the corresponding fluorescence ratio (targeted-NP vs. isotype-NP) from flow-cytometry experiments with the cell lines used to generate the various tumor xenografts. All values in the figures are presented as mean ± standard deviation. R > 0.95. Reproduced with permission from [12] © Biomedical Optics Express (2015).