Hyperspectral molecular imaging of multiple receptors using immunolabeled plasmonic nanoparticles

Abstract

This work presents simultaneous imaging and detection of three different cell receptors using three types of plasmonic nanoparticles (NPs). The size, shape, and composition-dependent scattering profiles of these NPs allow for a system of multiple distinct molecular markers using a single optical source. With this goal in mind, tags consisting of anti-epidermal growth factor receptor gold nanorods, anti-insulin-like growth factor 1-R silver nanospheres, and human epidermal growth factor receptor 2Ab gold nanospheres were developed to monitor the expression of receptors commonly overexpressed by cancer cells. These labels were chosen because they scatter strongly in distinct spectral windows. A hyperspectral darkfield microspectroscopy system was developed to record the scattering spectra of cells labeled with these molecular tags. Simultaneous monitoring of multiple tags may lead to applications such as profiling of cell line immunophenotype and investigation of receptor signaling pathways. Single, dual, and triple tag experiments were performed to analyze NP tag specificity as well as their interactions. Distinct resonance peaks were observed in these studies, showing the ability to characterize cell lines using conjugated NPs. However, interpreting shifts in these peaks due to changes in a cellular dielectric environment may be complicated by plasmon coupling between NPs bound to proximal receptors and other coupling mechanisms due to the receptors themselves.

Figure 1

(a) FACS results for A549 cells indicating top 5.18% of HER-2 expressers; (b) FACS results for A549 cells indicating top 13.17% of IGF-1R expressers.

Figure 2

(a) Hyperspectral darkfield microscopy system for analysis of live cells in culture. The system is comprised of a Zeiss inverted microscope, Cascade:650 imaging CCD, Fianium SC450-2 supercontinuum light source, and Crystal Technologies acousto-optic tunable filter. (b) Spectral output of the AOTF at various wavelengths.

Figure 3

(a) Representative image and (b) scattering spectrum of a single MDA-MB-468 cell bound with anti-EGFR gold nanorods. (Scale bar = 10 μm.) (c) Distribution of peak scattering peak wavelengths of MDA-MB-468 cells (N = 150) bound with anti-EGFR gold nanorods (67.1 ± 6.1 × 32.0 ± 8.9 nm). Distribution fit has peak wavelength of 664.0 ± 9.3 nm.

Figure 4

(a) Representative image and (b) spectrum of single A549/IGF-1R cell bound with anti-IGF-1R immunolabeled silver nanospheres. (Scale bar = 10 μm.) (c) Distribution of peak scattering wavelength of A549/IGF-1R cells bound with anti-IGF-1R 100 nm silver nanospheres. Distribution fit has peak wavelength of 520.8 ± 11.3 nm (N = 102).

Figure 5

(a) Representative image and (b) spectrum of a single SK-BR-3 cell bound with HER-2 Ab labeled 60 nm gold nanospheres. (Scale bar = 10 μm.) Note that plasmonic coupling causes a redshift in the scattered light. (c) Distribution of peak scattering wavelength of SK-BR-3 cells bound with HER-2 Ab labeled 60 nm gold nanospheres. Distribution fit has peak wavelength of 587.0 ± 11.9 nm (N = 122). Adapted from Ref. .

Figure 6

(a) Representative image and (b) spectrum of a single A549 / HER-2 cell exposed to anti-EGFR nanorods and HER-2 nanospheres consecutively. (Scale bar = 10 μm.) (c) Distribution of peak scattering from A549 / HER-2 cells exposed to anti-EGFR gold nanorods and HER-2 Ab nanospheres consecutively.

Figure 7

(a) Representative image and (b) spectrum of a single A549 cell exposed to anti-EGFR nanorods and anti-IGF-1R nanospheres consecutively. (Scale bar = 10 μm.)

Figure 8

Distribution of peak scattering from A549 cells exposed to anti-EGFR gold nanorods and anti-IGF-1R nanospheres (a) consecutively, and (b) in reverse order. A blueshift of 41.4 nm is obererved in the peak scattering wavelength of the anti-EGFR label after reversing the order. Similarly, a redshift of 2.6 nm is observed for the anti-IGF-1R label after reversing the order.

Figure 9

(a) Representative image and (b) spectrum of a single A549/HER-2 cell exposed to HER-2 Ab nanorods and anti-IGF-1R nanospheres consecutively. (Scale bar = 10 μm.)

Figure 10

(a) Representative image and (b) spectrum of a single A549 / HER-2 cell exposed to anti-EGFR nanorods, anti-IGF-1R nanospheres, and HER-2 Ab nanospheres consecutively. (Scale bar = 10 μm.) (c) Distribution of peak scattering from A549 / HER-2 cells exposed to anti-EGFR nanorods, anti-IGF-1R nanospheres, and HER-2 Ab nanospheres consecutively.

Figure 11

(a) Representative image and (b) spectrum of a single A549 / HER-2 cell exposed to anti-EGFR nanorods, anti-IGF-1R nanospheres, and HER-2 Ab nanospheres consecutively. (Scale bar = 10 μm.)