Multiplexed Optical Imaging of Tumor-Directed Nanoparticles: A Review of Imaging Systems and Approaches

Abstract

In recent decades, various classes of nanoparticles have been developed for optical imaging of cancers. Many of these nanoparticles are designed to specifically target tumor sites, and specific cancer biomarkers, to facilitate the visualization of tumors. However, one challenge for accurate detection of tumors is that the molecular profiles of most cancers vary greatly between patients as well as spatially and temporally within a single tumor mass. To overcome this challenge, certain nanoparticles and imaging systems have been developed to enable multiplexed imaging of large panels of cancer biomarkers. Multiplexed molecular imaging can potentially enable sensitive tumor detection, precise delineation of tumors during interventional procedures, and the prediction/monitoring of therapy response. In this review, we summarize recent advances in systems that have been developed for the imaging of optical nanoparticles that can be heavily multiplexed, which include surface-enhanced Raman-scattering nanoparticles (SERS NPs) and quantum dots (QDs). In addition to surveying the optical properties of these various types of nanoparticles, and the most-popular multiplexed imaging approaches that have been employed, representative preclinical and clinical imaging studies are also highlighted.

Keywords: Raman spectroscopy; hyperspectral imaging; multiplexed molecular imaging; preclinical study; surgical guidance.; tissue diagnosis.

Figure 1

Representative fluorescent probes and Raman-based nanoparticles as well as their emission spectra. The cartoons on the upper row only show one example of each probe type, and are not strictly drawn to scale. For the spectra shown on the bottom row, the dashed lines denote excitation spectra, and the solid lines denote emission/scattering spectra. The scattering spectra of the SERS NPs and carbon nanotubes are generated with 785-nm excitation. Since they utilize a scattering process, these nanoparticles can be excited across a broad range of wavelengths from the UV to NIR, but with varying efficiencies.

Figure 2

Schematic of the linear unmixing of signals from multiple fluorophores. The colored circles represent beads labeled with three different fluorophores. The emission spectra of the fluorophores overlap and cannot be well separated with bandpass filters. However, by using a linear unmixing algorithm based on the known signal contributions (the reference spectra) of each fluorophore in each detection channel, the overlapped signals can be clearly separated. The composite image is color-coded based on fluorophore type/flavor.

Figure 3

Schematic of optical imaging approaches for different imaging probes. The solid lines represent widely used approaches, and the dash lines represent rarely used or potential approaches.

Figure 4

Typical (hyper)spectral imaging approaches. (A) Point scan. (B) Line scan (i.e. “pushbroom”). (C) Wavelength scan. (D) Snapshot.

Figure 5

Raman-enabled molecular imaging of a human breast tissue specimen stained with a 5-flavor SERS NP mixture (EGFR-, HER2-, CD24-, CD44-, and isotype-NPs, 150 pM/flavor) - a comparison of spectral imaging and unmixing using 1024 and binned (32, 16 and 8) spectral channels. The mixture of SERS NPs was topically applied on fresh tissue surfaces for 5 min, followed by a 10-s rinse-removal step in PBS and raster-scanned spectral imaging with a 785-nm laser (3 min). (A) Ratiometric images of a human breast tissue specimen. From top to bottom, the rows display ratiometric images of EGFR/isotype-NP, HER2/isotype-NP, CD24/isotype-NP and CD44/isotype-NP. From left to right, the columns display ratiometric images obtained with a decreasing number of spectral channels. (B) A photograph of the tissue specimen. (C) H&E histology of the specimen, with higher magnification views of fat (left), normal breast tissue (middle), and tumor (right). Unlabeled scale bars represent 200 μm. (D) Average error (%) in the measured NP ratios when using spectral compression in comparison to the gold-standard images (1024 spectral channels). The error bars represent the standard deviation amongst all pixels in the image. These results showed that a low spectral resolution of 4 nm (32 bins) or 8 nm (16 bins) may still allow accurate unmixing of 5 SERS NP flavors, suggesting the potential of using advanced detector arrays (e.g. 16/32-channel PMT/APD array) to achieve high imaging speed. Reproduced with permission from .

Figure 6

Multispectral scanning fiber endoscope. (A) Optical design. RGB laser excitation (440, 532, and 635 nm) is delivered into a single-mode optical fiber that is scanned in a spiral pattern by a piezo tube actuator and focused onto the tissue (illumination plane) by a lens assembly. Fluorescence is collected by a ring of 12 collection fibers mounted around the periphery of the scanner housing, protected by an outer sheath. (B) Fluorescence detection. Reflectance from RGB laser excitation is removed using a combination of longpass (λ_LP = 450 nm) and notch (λ_N1 = 532 nm and λ_N2 = 632.8 nm) filters. Fluorescence is deflected into individual RGB channels using dichroic mirrors DM1 (λ_C = 460 nm) and DM2 (λ_C = 550 nm) and an additive dichroic filter set (λ_R, λ_G, and λ_B) prior to detection with PMTs. Reproduced with permission from .

Figure 7

Schematic of a customized spectral-imaging endoscope to detect multiplexed SERS NPs within the esophagus of a rat. The fiber-bundle imaging probe rotates between ± 180° as it is slowly pulled out of the esophagus. The inset on the bottom left is a zoom-in rendering of the prism and fiber-bundle imaging probe (distal end) within a glass guide tube and rat esophagus. Reproduced with permission from .

Figure 8

Integrin-targeted SERRS nanoparticles enable the detection of bulk and infiltrative glioblastomas. (A) Conceptual figure outlining integrin-based detection and delineation of glioblastoma with cRGDyK-conjugated SERRS nanoparticles. Due to the enhanced permeability and retention (EPR) effect of the GBMs, accumulation of the non-targeted RAD-SERRS nanoparticles (green) was limited to the bulk tumor, while the RGD-SERRS probe (red) could be detected in both the bulk tumor and the infiltrating tumor cells due to targeting of overexpressed integrin receptors. (B) SERRS spectra and (C) transmission electron micrographs (TEM) of the RGD-SERRS nanoparticles (red) and the non-targeted control RAD-SERRS nanoparticles (green). Scale bar = 100 nm. (D) Integrin-targeted SERRS nanoparticles enable the detection of bulk and infiltrative glioblastomas. GBM-bearing animals were co-injected with equimolar amounts of RGD-SERRS (targeted; red) and RAD-SERRS (non-targeted; green) nanoparticles. Multiplexed Raman imaging was performed on a coronal brain section (thickness 1 mm) in a paraffin block generated from a representative GBM-bearing mouse. Both RGD-SERRS (red) and RAD-SERRS (green) nanoparticles were detected in the bulk tumor located in the right hemisphere (OLIG2-positive). RGD-SERRS nanoparticles outlined the true tumor extent markedly better than the RAD-SERRS nanoparticles. (E) Sequential sections cut from the Raman-imaged paraffin block processed with H&E staining and immunohistochemistry of integrin β3 (α-ITGB3, target of the RGD-SERRS nanoparticles), OLIG2 (α-OLIG2), and polyethylene glycol linker (α-PEG, nanoparticle presence), respectively. (F) Pearson correlation analysis of the RGD-SERRS positive areas and OLIG-2 staining (n=7) was performed and a strong correlation (R=0.84) was found. Reproduced with permission from .

Figure 9

Multiplexed molecular imaging of freshly excised breast tissues with SERS NPs for guiding breast-conserving surgery. Each tissue specimen was stained with an equimolar mixture of HER2-NPs, EGFR-NPs, CD44-NPs, CD24-NPs and isotype-NPs (5 min), followed by a quick rinse in PBS (10 s) and raster-scanned imaging (< 3 min) to simultaneously quantify the expression of four breast cancer biomarkers: EGFR, HER2, CD44 and CD24. (A) Photograph of a human breast tumor and a normal tissue specimen from one patient. (B) Ratiometric images of EGFR-NPs vs. isotype-NPs, HER2-NPs vs. isotype-NPs, CD44-NPs vs. isotype-NPs and CD24-NPs. (C) Validation data: H&E and IHC for EGFR, HER2, CD44 and CD24. Unlabeled scale bars represent 200 μm. (D) Cumulative results from multiple regions of interest from a total of 5 patient specimens: measured NP ratios on IHC-validated biomarker-negative and biomarker-positive tissue regions. Each data point in the plots is the average ratio from one region of interest. Reproduced with permission from .

Figure 10

Multiplexed molecular profiling of FFPE tissue sections using QDs. Different patterns of nuclear, cytoplasmic and cell membrane fluorescent signals were detected by microscopy (left panels, pseudo-color) and expression of these biomarkers was quantified by wavelength-resolved spectroscopy (right panels). The emission spectra were resolved into individual channels and compensated for the differential brightness between different colors. Scale bar represents 20 µm. Reproduced with permission from .

Figure 11

QD-based IHC for molecular pathology. (A, B) Multispectral imaging of 4 QDs for detection of rare Hodgkin's and Reed-Sternberg (HRS) tumor cells in Hodgkin's lymphoma. The images show HRS malignant cells and infiltrating immune cells on lymph node tissue specimens. The HRS cells (arrows) exhibited a characteristic staining pattern: membrane staining (CD30 positive, red), Golgi staining (CD15 positive, white), and nuclear staining (Pax5 positive, blue). Staining patterns were clearly distinct from infiltrating B cells (blue nuclear staining) and T cells (green membrane staining). The scale bar in (A) represents 100 μm; the scale bar in (B) represents 10 μm. Reproduced with permission from . Copyright (2010) American Chemical Society. (C, D) QD multiplexed staining of four protein biomarkers in human prostatectomy specimens highlighting cellular and glandular heterogeneity and tumor progression. The four biomarkers are E-cadherin (green), cytokeratin HMW (white), p63 (red) and AMACR (blue). (C) Largely benign prostate gland with a single malignant cell (arrow) in the luminal layer, as determined by positive AMACR staining. (D) Completely malignant gland, as determined by intense AMACR staining and absent basal cell cytokeratin and p63 signals. The scale bar in (C, D) represents 10 μm. Reproduced with permission from . Copyright (2010) American Chemical Society.