Two-photon imaging is a technique that offers a powerful means of diagnosing epithelial cancers early because it allows noninvasive imaging of subcellular features hundreds of micrometers deep in tissue. Imaging endogenous fluorophores, researchers have shown that they can distinguish cancerous and precancerous cells as deep as 40 μm. By incorporating fluorescent contrast agents, they also have tracked additional biomolecular signatures that can help identify cancer.Investigators have therefore devoted considerable effort to the use of organic fluorophores and luminescent contrast agents such as quantum dots and metallic nanoparticles. Quantum dots have stirred interest because they offer much larger two-photon action cross sections than organic fluorophores. They are not ideal for in vivo applications, however, because they often contain cytotoxic heavy metals. Gold nanoparticles have large two-photon action cross sections and are biocompatible, offering an attractive alternative for two-photon imaging.In an April Nano Letters paper, researchers with the University of Texas at Austin and the University of Texas M.D. Anderson Cancer Center in Houston reported a study in which they used gold nanorods as bright contrast agents for two-photon luminescence imaging of cancer cells. “We showed that the nanorods are much brighter than autofluorescence, for example — three to four orders of magnitude brighter under the same illumination conditions,” said Adela Ben-Yakar, the principal investigator of the study.Single-photon-induced luminescence was first described in 1969. Researchers later found that they could increase luminescence efficiency by using roughened as opposed to smooth metal surfaces. They also noted that roughened surfaces enable two-photon-induced luminescence — most likely because of efficient coupling between particular frequencies of light and surface plasmons.Gold nanorods offer highly efficient single- and two-photon-induced luminescence, possibly because they can sustain resonating surface plasmons with little damping. Also, users can tune the longitudinal plasmonic resonance to the near-infrared, the region most suited to measurement of biological tissue. Researchers have demonstrated that gold nanorods used as bright contrast agents are three to four orders of magnitude brighter than autofluorescence when employed for two-photon luminescence imaging. Shown here are two-photon images of cancer cells: an autofluorescence image of unlabeled cells (a), an image of nanorod-labeled cells (b) and an image of nonspecifically labeled cells (c). Imaging of unlabeled cells required 9 mW of excitation power (Pin ) to obtain the same signal level acquired with only 140 μW with nanorod-labeled cells. Images reprinted with permission of Nano Letters.To test the efficacy of gold nanorods for two-photon luminescence imaging of cancer cells, the University of Texas researchers imaged nanorod-labeled oral cancer cells embedded in a collagen matrix, mimicking the cells in tissue. To this end, they used a home-built inverted multiphoton microscope with a femtosecond Ti:sapphire laser made by Spectra-Physics of Mountain View, Calif., as an excitation source. At 760 nm, the pulse length provided by the laser was estimated to be 250 fs at the sample. Using a set of galvanometric scanning mirrors, they raster-scanned the laser into the back aperture of a 64x, 1.4-NA Zeiss oil-immersion objective. Light emitted from the sample was epicollected, reflected by a cold mirror and, after passing through a laser filter, detected with a cooled Hamamatsu GaAsP photomultiplier tube. Images of the cells were reconstructed in real time using a National Instruments data acquisition card.The 145-μm field of view was scanned into a 512 × 512-pixel image at a rate of 1.5 fps. Based on the optics used and the response of the photomultiplier tube, the system collected emission light in the range of 400 to 700 nm.3-D imagingThe gold nanorods let the researchers image oral cancer cells in the three-dimensional tissue phantom down to 75 μm deep, not quite as deep as they would have liked. “We were limited by our objective lens — an oil-immersion lens with a short working distance,” Ben-Yakar explained. To address this issue, they plan to build an upright microscope that will permit them to use a water-immersion lens with a working distance of 2 mm. With this, they should easily achieve 200 μm, she said. Their goal is 500 μm. Researchers performed two-photon imaging of cancer cells embedded in a phantom, at increasing depths, with autofluorescence imaging (a) and imaging of nanorod-labeled cells (b). Again, imaging of nanorod-labeled cells required considerably less excitation power.The investigators noted a variety of advantages to using gold nanorods for two-photon luminescence imaging, in addition to the strong signal and biocompatibility: resistance to photobleaching, chemical stability, ease of synthesis and simplicity of conjugation chemistry. Thus, the nanorods could serve as effective contrast agents for two-photon imaging of epithelial cancer.The nanorods also could contribute to cancer therapy. The researchers are planning to incorporate two-photon imaging with gold nanorods in another project in which they will perform surgery using plasmonic ablation. In this technique the nanorod acts as a nanolens to enhance the light around the particle, Ben-Yakar said, eliminating the need for a tightly focused beam without sacrificing nanoscale resolution. “We will use this process to ablate cancer cells with much more precision and much less energy than is possible with thermal ablation; we’ll be able to image deep and then remove the cancer cells without heating of the surrounding tissue,” she said.