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.
This work presents simultaneous imaging and detection of three types of cell receptors using three types of
plasmonic nanoparticles. The size, shape, and composition-dependent scattering profiles of these particles allow for a
system of multiple distinct molecular markers using a single optical source. With this goal in mind, a system of tags
consisting of anti-EGFR gold nanorods, anti-IGF1R silver nanospheres, and anti-HER-2 gold nanospheres was
developed for monitoring the expression of three commonly overexpressed receptors in cancer cells. These labels were
chosen because they each scatter strongly in a distinct spectral window. A hyperspectral dark-field microscope was
developed to record the scattering spectra of cells labeled with these molecular tags. The ability to monitor multiple tags
simultaneously may lead to applications such as profiling the immunophenotype of cell lines and gaining better
knowledge of receptor signaling pathways. Single, dual, and triple tag experiments were performed to analyze the
specificity of the nanoparticle tags as well as their effect on one another. While distinct resonance peaks in these studies
show the ability to characterize cell lines using conjugated nanoparticles, shifts in these peaks also indicate changes in
the cellular dielectric environment which may not be distinct from plasmon coupling between nanoparticles bound to proximal receptors.
Molecular imaging is a powerful tool for investigating disease processes and potential therapies
in both in vivo and in vitro systems. However, high resolution molecular imaging has been
limited to relatively shallow penetration depths that can be accessed with microscopy. Optical
coherence tomography (OCT) is an optical analogue to ultrasound with relatively good
penetration depth (1-2 mm) and resolution (~1-10 μm). We have developed and characterized
photothermal OCT as a molecular contrast mechanism that allows for high resolution molecular
imaging at deeper penetration depths than microscopy. Our photothermal system consists of an
amplitude-modulated heating beam that spatially overlaps with the focused spot of the sample
arm of a spectral-domain OCT microscope. Validation experiments in tissue-like phantoms
containing gold nanospheres that absorb at 532 nm revealed a sensitivity of 14 parts per million
nanospheres (weight/weight) in a tissue-like environment. The nanospheres were then
conjugated to anti-EGFR, and molecular targeting was confirmed in cells that over-express
EGFR (MDA-MB-468) and cells that express low levels of EGFR (MDA-MB-435). Molecular
imaging in three-dimensional tissue constructs was confirmed with a significantly lower
photothermal signal (p<0.0001) from the constructs composed of cells that express low levels of
EGFR compared to the over-expressing cell constructs (300% signal increase). This technique
could potentially augment confocal and multiphoton microscopy as a method for deep-tissue,
depth-resolved molecular imaging with relatively high resolution and target sensitivity, without
photobleaching or cytotoxicity.
Epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER2)
contribute to the regulation of cell proliferation, and when jointly over-expressed are associated with
several types of cancer. The ability to monitor both receptors simultaneously results in a more accurate
indicator of degree of cancerous activity than either receptor alone. Plasmonic nanoparticles (NPs) show
promise as a potential EGFR and HER2 biomarker over alternatives such as fluorophores and quantum
dots, which are limited by their cytotoxicity and photobleaching. To observe immunolabeled NPs bound to
receptor-expressing cells, our past experiments were conducted using a novel optical darkfield
microspectroscopy system. We implemented an epi-illumination darkfield broadband light train, which
allows for darkfield analysis of live cells in culture with enhanced NP contrast. Under this setup,
molecularly specific binding of NPs immunolabeled with anti-EGFR was confirmed.
We have since adapted our darkfield setup, which previously only obtained spectral information from a
line imaging spectrometer, to incorporate hyperspectral imaging capabilities, allowing widefield data
acquisition within seconds. The new system has been validated through observation of shifts in the peak
wavelength of scattering by gold NPs on silanated cover glasses using several immersion media. Peak
resonant scattering wavelengths match well with that predicted by Mie theory. We will further demonstrate
the potential of the system with simultaneous molecular imaging of multiple receptors in vitro using labeled
EGFR+/HER2+ SK-BR-3 human breast cancer cells with anti-EGFR immunolabeled gold nanospheres and
anti-HER2 immunolabeled gold nanorods, with each scattering in different spectral windows. Additional
trials will be performed to demonstrate molecularly specific binding using EGFR+/HER2- MDA-MB-468
and HER2+/EGFR- MDA-MB-453 breast cancer cells.
We present a study using plasmonic nanoparticles (NPs) to image epidermal growth factor receptor (EGFR) in live cells. Through detailed analysis of the NP scattering spectra, we determine the intracellular refractive index (RI) within attoliter volumes inside of the living cells. Molecular imaging is demonstrated using anti-EGFR labeled gold nanospheres delivered to cancer cells that overexpress EGFR, with targeted binding confirmed by appropriate control experiments. RI determination is achieved by measurement of the bound NPs' scattering spectra, acquired using a precision dark-field microspectroscopy system and through careful characterization of the NP properties throughout the immuno-labeling procedure. To demonstrate the effect of receptor-mediated uptake, the data are compared to similar spectral measurements using antibody-free NPs, taken up by the cells through nonspecific mechanisms. In these experiments, NP aggregation introduces interparticle effects in the scattering spectra, suggesting that EGFR-mediated internalization of NPs may provide an advantage for maintaining NP isolation upon uptake. The results of this study show the potential utility of dark-field microspectroscopy and labeled NPs for live cell imaging. By demonstrating RI sensitivity over nanometer length scales, this study also presents a potential new avenue for assessing the structure and dynamics of live cells.
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