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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 x 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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