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Gold nanoparticles can be used as contrast agents for bio-imaging applications. Here we studied multi-photon luminescence (MPL) of gold nanorods (GNRs), under the excitation of femtosecond (fs) lasers. GNRs functionalized with polyethylene glycol (PEG) molecules have high chemical and optical stability, and can be used as multi-photon luminescent nanoprobes for deep in vivo imaging of live animals. We have found that the depth of in vivo imaging is dependent upon the transmission and focal capability of the excitation light interacting with the GNRs. Our study focused on the comparison of MPL from GNRs with two different aspect ratios, as well as their ex vivo and in vivo imaging effects under 760 nm and 1000 nm excitation, respectively. Both of these wavelengths were located at an optically transparent window of biological tissue (700-1000 nm). PEGylated GNRs, which were intravenously injected into mice via the tail vein and accumulated in major organs and tumor tissue, showed high image contrast due to distinct three-photon luminescence (3PL) signals upon irradiation of a 1000 nm fs laser. Concerning in vivo mouse brain imaging, the 3PL imaging depth of GNRs under 1000 nm fs excitation could reach 600 μm, which was approximately 170 μm deeper than the two-photon luminescence (2PL) imaging depth of GNRs with a fs excitation of 760 nm.

Three-Photon Luminescence of Gold Nanorods and Its Applications for High Contrast Tissue and Deep In Vivo Brain Imaging