Elasto-optical refractive index modulation due to photoacoustic initial pressure transients produced significant reflection of a probe beam when the absorbing interface had an appreciable refractive index difference. This effect was harnessed in a new form of non-contact optical resolution photoacoustic microscopy called photoacoustic remote sensing microscopy. A non-interferometric system architecture with a low-coherence probe beam precludes detection of surface oscillations and other phase-modulation phenomenon. The probe beam was confocal with a scanned excitation beam to ensure detection of initial pressure-induced intensity reflections at the subsurface origin where pressures are largest. Phantom studies confirmed signal dependence on optical absorption, index contrast and excitation fluence. In vivo imaging of superficial microvasculature and melanoma tumors was demonstrated with ~2.7±0.5 μm lateral resolution.

Reporter genes are useful scientific tools for analyzing promoter activity, transfection efficiency, and cell migration. The current study has validated the use of tyrosinase (involved in melanin production) as a dual reporter gene for magnetic resonance and photoacoustic imaging. MCF-7 cells expressing tyrosinase appear brown due to melanin. Magnetic resonance imaging of tyrosinase-expressing MCF-7 cells in 300 μL plastic tubes displayed a 34 to 40% reduction in T1 compared to normal MCF-7 cells when cells were incubated with 250 μM ferric citrate. Photoacoustic imaging of tyrosinase-expressing MCF-7 cells in 700 μm plastic tubes displayed a 20 to 57-fold increase in photoacoustic signal compared to normal MCF-7 cells. The photoacoustic signal from tyrosinase-expressing MCF-7 cells was significantly greater than blood at 650 nm, suggesting that tyrosinase-expressing cells can be differentiated from the vasculature with in vivo photoacoustic imaging. The imaging results suggest that tyrosinase is a useful reporter gene for both magnetic resonance and photoacoustic imaging.

3Ј-Deoxy-3Ј-fluorothymidine (FLT) is a positron emission tomography (PET) tracer used to identify proliferating tumor cells. The purpose of this study was to characterize FLT transport by human nucleoside transporters (hNTs) and to determine the role of hNTs for FLT uptake in various human cancer cell lines.

A novel class of all-organic nanoscale porphyrin nanodroplet agents is presented which is suitable for multimodality ultrasound and photoacoustic molecular imaging. Previous multimodality photoacoustic-ultrasound agents are either not organic, or not yet demonstrated to exhibit enhanced accumulation in leaky tumor vasculature, perhaps because of large diameters. In the current study, porphyrin nanodroplets are created with a mean diameter of 185 nm which is small enough to exhibit the enhanced permeability and retention effect. Porphyrin within the nanodroplet shell has strong optical absorption at 705 nm with an estimated molar extinction coefficient >5 × 10(9) m(-1) cm(-1) , allowing both ultrasound and photoacoustic contrast in the same nanoparticle using all organic materials. The potential of nanodroplets is that they may be phase-changed into microbubbles using high pressure ultrasound, providing ultrasound contrast with single-bubble sensitivity. Multispectral photoacoustic imaging allows visualization of nanodroplets when injected intratumorally in an HT1080 tumor in the chorioallantoic membrane of a chicken embryo. Intravital microscopy imaging of Hep3-GFP and HT1080-GFP tumors in chicken embryos determines that nanodroplets accumulated throughout or at the periphery of tumors, suggesting that porphyrin nanodroplets may be useful for enhancing the visualization of tumors with ultrasound and/or photoacoustic imaging.

Photoacoustic imaging is an emerging hybrid imaging technology capable of breaking through resolution limits of pure optical imaging technologies imposed by optical-scattering to provide fine-resolution optical contrast information in deep tissues. We demonstrate the ability of multi-wavelength photoacoustic imaging to estimate relative gene expression distributions using an inducible expression system and co-register images with hemoglobin oxygen saturation estimates and micro-ultrasound data. Tyrosinase, the rate-limiting enzyme in melanin production, is used as a reporter gene owing to its strong optical absorption and enzymatic amplification mechanism. Tetracycline-inducible melanin expression is turned on via doxycycline treatment in vivo. Serial multi-wavelength imaging reveals very low estimated melanin expression in tumors prior to doxycycline treatment or in tumors with no tyrosinase gene present, but strong signals after melanin induction in tumors tagged with the tyrosinase reporter. The combination of new inducible reporters and high-resolution photoacoustic and micro-ultrasound technology is poised to bring a new dimension to the study of gene expression in vivo.

18 F-39-Deoxy-39-fluorothymidine ( 18 F-FLT) is a PET tracer that accumulates in proliferating tissues. The current study was undertaken to determine whether equilibrative nucleoside transporter 1 (ENT1) is important for 18 F-FLT uptake in normal tissues and tumors. Methods: ENT1-knockout (ENT1 2/2 ) mice were generated and compared with wild-type (ENT1 1/1 ) mice using small-animal 18 F-FLT PET. In addition, ENT1 1/1 mice were also injected with the ENT1 inhibitor nitrobenzylmercaptopurine ribonucleoside phosphate (NBMPR-P) at 1 h before radiotracer injection, followed by 18 F-FLT small-animal PET. Tissues of interest were analyzed for thymidine kinase 1 and nucleoside transporters by immunoblotting and immunohistochemistry, respectively, and plasma thymidine levels were analyzed by liquid chromatography-mass spectrometry. Human lung carcinoma A549 cells were stably transfected with pSUPER-producing short-hairpin RNA against human ENT1 (hENT1) or a scrambled sequence with no homology to mammalian genes (A549-pSUPER-hENT1 and A549-pSUPER-SC, respectively). Cultured transfected cells were characterized for hENT1 transcript levels and 18 F-FLT uptake using real-time polymerase chain reaction and 3 H-FLT uptake assays, respectively. Transfected A549 cells were grown as xenograft tumors in NIH-III mice, which were analyzed by 18 F-FLT small-animal PET. Results: Compared with noninjected ENT1 1/1 mice, ENT1 1/1 mice injected with NBMPR-P and ENT1 2/2 mice displayed a reduced percentage injected dose per gram (%ID/g) for 18 F-FLT in the blood (84 and 81%, respectively) and an increased %ID/g for 18 F-FLT in the spleen (188 and 469%, respectively) and bone marrow (266 and 453%, respectively). ENT1 2/2 mice displayed 1.65-fold greater plasma thymidine levels than did ENT1 1/1 mice. Spleen tissue from ENT1 1/1 and ENT1 2/2 mice displayed similar thymidine kinase 1 protein levels and significant concentrative nucleoside transporter 1 and 3 staining. Compared with A549-pSUPER-SC cells, A549-pSUPER-hENT1 cells displayed 0.45-fold hENT1 transcript levels and 0.68-fold 3 H-FLT uptake. Compared with A549-pSUPER-SC xenograft tumors, A549-pSUPER-hENT1 xenograft tumors displayed 0.76-fold %ID/g values (ex vivo g-counts) and 0.65-fold maximum standardized uptake values (PET image analysis) for 18 F-FLT uptake at 1 h after tracer injection. Conclusion: Loss of ENT1 activity significantly affected 18 F-FLT biodistribution in mice and 18 F-FLT uptake in xenograft tumors, suggesting that nucleoside transporters are important mediators of 18 F-FLT uptake in normal and transformed cells.

Abstract. While more than 90% of cancer deaths are due to metastases, our ability to detect circulating tumor cells (CTCs) is limited by low numbers of these cells in the blood and factors confounding specificity of detection. We propose a magnetic enrichment and detection technique for detecting CTCs with high specificity. We targeted both magnetic and surface-enhanced Raman scattering (SERS) nanoparticles to cancer cells. Only cells that are dual-labeled with both kinds of nanoparticles demonstrate an increasing SERS signal over time due to magnetic trapping.

Metastasis is the most lethal aspect of cancer, yet current therapeutic strategies do not target its key rate-limiting steps. We have previously shown that the entry of cancer cells into the blood stream, or intravasation, is highly dependent upon in vivo cancer cell motility, making it an attractive therapeutic target. To systemically identify genes required for tumor cell motility in an in vivo tumor microenvironment, we established a novel quantitative in vivo screening platform based on intravital imaging of human cancer metastasis in ex ovo avian embryos. Utilizing this platform to screen a genome-wide shRNA library, we identified a panel of novel genes whose function is required for productive cancer cell motility in vivo, and whose expression is closely associated with metastatic risk in human cancers. The RNAi-mediated inhibition of these gene targets resulted in a nearly total (>99.5%) block of spontaneous cancer metastasis in vivo.