We demonstrate the use of gold nanorods as molecularly targeted contrast agents for two-photon luminescence (TPL) imaging of cancerous cells 150 μm deep inside a tissue phantom. We synthesized gold nanorods of 50 nm × 15 nm size with a longitudinal surface plasmon resonance of 760 nm. Gold nanorods were conjugated to antibodies against epidermal growth factor receptor (EGFR) and labeled to A431 human epithelial skin cancer cells in a collagen matrix tissue phantom. Using a 1.4 NA oil immersion objective lens, we found that excitation power needed for similar emission intensity in TPL imaging of labeled cells was up to 64 times less than that needed for two-photon autofluorescence (TPAF) imaging of unlabeled cells, which would correspond to a more than 4,000 times increase in emission intensity under equal excitation energy. However, the aberrations due to refractive index mismatch of the immersion oil and the sample limit imaging depth to 75 μm. Using a 0.95 NA water immersion objective lens, we observe robust two-photon emission signal from gold nanorods in the tissue phantoms from at depths of up to 150 μm. Furthermore, the increase in excitation energy required to maintain a constant emission signal intensity as imaging depth was increased was the same in both labeled and unlabeled phantom, suggesting that at the concentrations used, the addition of gold nanorods did not appreciably increase the bulk scattering coefficient of the sample. The remarkable TPL brightness of gold nanorods in comparison to TPAF signal makes them an attractive contrast agent for early detection of cutaneous melanoma.
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