Gold nanorods excited at 830 nm on a far-field laser-scanning microscope produced strong two-photon luminescence (TPL) intensities, with a cos 4 dependence on the incident polarization. The TPL excitation spectrum can be superimposed onto the longitudinal plasmon band, indicating a plasmon-enhanced two-photon absorption cross section. The TPL signal from a single nanorod is 58 times that of the two-photon fluorescence signal from a single rhodamine molecule. The application of gold nanorods as TPL imaging agents is demonstrated by in vivo imaging of single nanorods flowing in mouse ear blood vessels.in vivo imaging ͉ plasmon resonance ͉ multiphoton ͉ nonlinear optics P hotoluminescence from noble metals was first reported in 1969 by Mooradian (1) and later observed as a broad background in surface-enhanced Raman scattering (2). Singlephoton luminescence from metals has been described as a three-step process as follows: (i) excitation of electrons from the d-to the sp-band to generate electron-hole pairs, (ii) scattering of electrons and holes on the picosecond timescale with partial energy transfer to the phonon lattice, and (iii) electron-hole recombination resulting in photon emission (1). Two-photon luminescence (TPL) was characterized by Boyd et al. (3) and is considered to be produced by a similar mechanism as singlephoton luminescence, but the relatively weak TPL signal can be amplified by several orders of magnitude when produced from roughened metal substrates. This amplification is due to a resonant coupling with localized surface plasmons, which are well known to enhance a variety of linear and nonlinear optical properties (4-9).Metal nanoparticles are also capable of photoluminescence, which has been shown to correlate strongly with their welldefined plasmon resonances (10-16). For example, Mohamed et al. (11) have observed that the quantum efficiency of singlephoton luminescence from gold nanorods is enhanced by a factor of Ͼ1 million under plasmon-resonant conditions. Plasmonresonant TPL is attractive for nonlinear optical imaging of biological samples with 3D spatial resolution (17). Gold nanorods are particularly appealing as TPL substrates because their longitudinal plasmon modes are resonant at near-infrared, where the absorption of water and biological molecules are minimized. Moreover, nanorods have larger local field enhancement factors than nanoparticles due to reduced plasmon damping (18). A scanning near-field optical microscopy study of TPL from single nanorods (diameter Ϸ 40 nm) has recently been reported by Imura et al. (16), who observed that the luminescence is greatest at their tips. However, further characterization of TPL from single gold nanorods is needed: the polarization dependence of TPL excitation and emission from nanorods has yet to be defined, as well as the relationship between TPL enhancement and the longitudinal and transverse plasmon modes. These studies can provide a deeper understanding of single-particle TPL and its potential application in nonlinear optical imag...

Folate‐conjugated gold nanorods targeted to tumor cell surfaces produced severe membrane damage upon near‐infrared irradiation. Photoinduced injury to the plasma membrane resulted in a rapid increase in intracellular calcium (shown in green) with subsequent disruption of the actin network, featured prominently by the formation of membrane blebs.

SummaryPlasmon-resonant gold nanorods, which have large absorption cross sections at near-infrared (NIR) frequencies, are excellent candidates as multifunctional agents for image-guided therapies based on localized hyperthermia. The controlled modification of the nanorods' surface chemistry is of critical importance, as issues of cell-specific targeting and nonspecific uptake must be addressed prior to clinical evaluation. Nanorods coated with CTAB (a cationic surfactant used in nanorod synthesis) are internalized within hours into KB cells by a nonspecific uptake pathway, whereas the careful removal of CTAB from nanorods functionalized with folate results in their accumulation on the cell surface over the same time interval. In either case, the nanorods render the tumor cells highly susceptible to photothermal damage when irradiated at the nanorods' longitudinal plasmon resonance, generating extensive blebbing of the cell membrane at laser fluences as low as 44 W/cm 2 .

Gold nanorods coated with cetyltrimethylammonium bromide (CTAB), a cationic micellar surfactant used in nanorod synthesis, were rapidly and irreversibly internalized by KB cells via a nonspecific uptake mechanism. Internalized nanorods near the cell surface were monitored by two-photon luminescence (TPL) microscopy and observed to migrate toward the nucleus with a quadratic rate of diffusion. The internalized nanorods were not excreted but formed permanent aggregates within the cells, which remained healthy and grew to confluence over a 5-day period. Nonspecific nanorod uptake could be greatly reduced by displacing the CTAB surfactant layer with chemisorptive surfactants, particularly by the conjugation of polyethylene glycol chains onto nanorods using in situ dithiocarbamate formation.

BackgroundLipid-rich tumours have been associated with increased cancer metastasis and aggressive clinical behaviours. Nonetheless, pathologists cannot classify lipid-rich tumours as a clinically distinctive form of carcinoma due to a lack of mechanistic understanding on the roles of lipids in cancer development.MethodsCoherent anti-Stokes Raman scattering (CARS) microscopy is employed to study cancer cell behaviours in excess lipid environments in vivo and in vitro. The impacts of a high fat diet on cancer development are evaluated in a Balb/c mice cancer model. Intravital flow cytometry and histology are employed to enumerate cancer cell escape to the bloodstream and metastasis to lung tissues, respectively. Cancer cell motility and tissue invasion capability are also evaluated in excess lipid environments.ResultsCARS imaging reveals intracellular lipid accumulation is induced by excess free fatty acids (FFAs). Excess FFAs incorporation onto cancer cell membrane induces membrane phase separation, reduces cell-cell contact, increases surface adhesion, and promotes tissue invasion. Increased plasma FFAs level and visceral adiposity are associated with early rise in circulating tumour cells and increased lung metastasis. Furthermore, CARS imaging reveals FFAs-induced lipid accumulation in primary, circulating, and metastasized cancer cells.ConclusionLipid-rich tumours are linked to cancer metastasis through FFAs-induced physical perturbations on cancer cell membrane. Most importantly, the revelation of lipid-rich circulating tumour cells suggests possible development of CARS intravital flow cytometry for label-free detection of early-stage cancer metastasis.