This study demonstrates the use of gold nanorods as bright contrast agents for two-photon luminescence (TPL) imaging of cancer cells in a three-dimensional tissue phantom down to 75 μm deep. The TPL intensity from gold nanorod-labeled cancer cells is three orders of magnitude brighter than the two-photon autofluorescence (TPAF) emission from unlabeled cells at 760 nm excitation. Gold nanorods are attractive for TPL imaging due to their strong signal, resistance to photobleaching, chemical stability, ease of synthesis, simplicity of conjugation chemistry, and biocompatibility. Cancer often begins as precancerous lesions in the epithelium, which can be as thick as 500 μm in human tissue. TPL imaging is a powerful technique for early diagnosis of epithelial cancers because it allows non-invasive imaging of sub-cellular features deep into tissue. TPL imaging can distinguish cancerous and precancerous cells from normal tissue down to 40 μm deep. By attaching a fluorescent contrast agent to a non-fluorescent target, TPL can monitor additional biomolecular signatures indicative of cancer. Gold nanorods exhibit highly efficient single- and two-photon induced luminescence, which may be due to their ability to sustain resonating surface plasmons with minimal damping. Their longitudinal plasmonic resonance can be tuned to near-infrared wavelengths, where biological tissue has relatively small extinction coefficients. These optical properties make gold nanorods attractive for biomedical imaging of highly scattered tissue. The study presents TPL images of gold nanorod-labeled cancer cells acquired up to 75 μm deep in a tissue phantom. By embedding cells in a collagen matrix, the performance of nanorods as contrast agents for deep-tissue imaging was explored. TPL imaging of labeled cancer cells could be performed using less than 60 times the laser excitation power needed for TPAF imaging from unlabeled cells, corresponding to a three-order-of-magnitude increase in emitted signal for equal excitation intensity. Gold nanorods were synthesized using a seed-mediated, surfactant-assisted growth method. They were functionalized with polystyrene sulfonate and anti-EGFR antibodies for molecular targeting. The nanorods were then used to label cancer cells, which were embedded in a collagen matrix for imaging. TPL imaging of nanorod-labeled cells showed bright rings indicative of EGFR labeling and endosomal uptake of EGF receptors. TPL images of cells treated with non-specifically conjugated nanorods showed agglomeration of contrast agent and little attachment to cell membranes. TPL imaging of nanorod-labeled cells required 64 times less power than TPAF imaging of unlabeled cells to achieve similar collected intensity. The study also tested the imaging ability of TPL deep into tissue by imaging cancer cells embedded in a collagenThis study demonstrates the use of gold nanorods as bright contrast agents for two-photon luminescence (TPL) imaging of cancer cells in a three-dimensional tissue phantom down to 75 μm deep. The TPL intensity from gold nanorod-labeled cancer cells is three orders of magnitude brighter than the two-photon autofluorescence (TPAF) emission from unlabeled cells at 760 nm excitation. Gold nanorods are attractive for TPL imaging due to their strong signal, resistance to photobleaching, chemical stability, ease of synthesis, simplicity of conjugation chemistry, and biocompatibility. Cancer often begins as precancerous lesions in the epithelium, which can be as thick as 500 μm in human tissue. TPL imaging is a powerful technique for early diagnosis of epithelial cancers because it allows non-invasive imaging of sub-cellular features deep into tissue. TPL imaging can distinguish cancerous and precancerous cells from normal tissue down to 40 μm deep. By attaching a fluorescent contrast agent to a non-fluorescent target, TPL can monitor additional biomolecular signatures indicative of cancer. Gold nanorods exhibit highly efficient single- and two-photon induced luminescence, which may be due to their ability to sustain resonating surface plasmons with minimal damping. Their longitudinal plasmonic resonance can be tuned to near-infrared wavelengths, where biological tissue has relatively small extinction coefficients. These optical properties make gold nanorods attractive for biomedical imaging of highly scattered tissue. The study presents TPL images of gold nanorod-labeled cancer cells acquired up to 75 μm deep in a tissue phantom. By embedding cells in a collagen matrix, the performance of nanorods as contrast agents for deep-tissue imaging was explored. TPL imaging of labeled cancer cells could be performed using less than 60 times the laser excitation power needed for TPAF imaging from unlabeled cells, corresponding to a three-order-of-magnitude increase in emitted signal for equal excitation intensity. Gold nanorods were synthesized using a seed-mediated, surfactant-assisted growth method. They were functionalized with polystyrene sulfonate and anti-EGFR antibodies for molecular targeting. The nanorods were then used to label cancer cells, which were embedded in a collagen matrix for imaging. TPL imaging of nanorod-labeled cells showed bright rings indicative of EGFR labeling and endosomal uptake of EGF receptors. TPL images of cells treated with non-specifically conjugated nanorods showed agglomeration of contrast agent and little attachment to cell membranes. TPL imaging of nanorod-labeled cells required 64 times less power than TPAF imaging of unlabeled cells to achieve similar collected intensity. The study also tested the imaging ability of TPL deep into tissue by imaging cancer cells embedded in a collagen