The number of cells in each well was counted by hematocytometer. melanoma patients is very high (~99%), but the rate drops to 40% after nodal metastases.1,2Thick melanomas (>4 mm) are typically associated with a high risk of nodal and distant metastases. Highly sensitive molecular imaging techniques including positron emission tomography (PET) and optical imaging have been developed for detecting early-stage melanomas.35However, PET requires on the use of radio-labeling materials, which can cause potential hazards to patients. Moreover, it suffers from low spatial resolution and high cost due to the need of additional anatomical information from magnetic resonance imaging (MRI) and/or X-ray computed tomography (CT). Since optical Azaperone imaging uses nonionizing radiation and is cost-effective, it has received much attention in molecular imaging.6However, conventional optical imaging tools are often limited by either shallow penetration depth (<1 GADD45A mm)7or poor spatial resolution.8Besides early detection, accurate delineation of the margins of a melanoma can significantly improve surgical removal of the primary tumor. High-frequency ultrasound has been applied preoperatively for this purpose,9but it cannot effectively resolve the margins of a melanoma and does not infiltrate cells. Additionally, accurate staging (describing cancer metastasis, typically with figures I to IV) of patients after nodal metastases is usually important for treatment planning.10Again, the current technique based on sentinel lymph node biopsy is ionizing and intraoperative, and thus poses postoperative complications. These limitations of the current techniques suggest a strong need for a single, highly sensitive, safe, economical, noninvasive, and high-resolution imaging technique with nonradioactive contrast brokers in early diagnosis of malignant melanomas, image-guided resection of melanoma boundaries, and accurate staging of melanoma patients. PAT is a hybrid biomedical imaging modality that offers both strong optical absorption contrast and high ultrasonic resolution (Supporting Information).11,12Since the spatial resolution beyond one optical transport imply free path (~1 mm) is determined by ultrasonic parameters, the maximum imaging depth and resolution of PAT are scalable when diffusive photons are available.1315By using a near-infrared (NIR) light, one can greatly increase the penetration depth of PAT because the optical absorption of hemoglobin and scattering of tissues are weak in this regime. However, it remains a challenge to assess melanomas using only morphological and functional PAT based on intrinsic contrasts such as tumor hypoxia and angiogenesis. Consequently, a tumor-specific, exogenous contrast agent is necessary for the assessment of early-stage melanomas, image-guided resection, and accurate staging by PAT, with high sensitivity and specificity. Organic dyes such as IRDye-800 and indocyanine Azaperone green have been used as contrast brokers for PAT,1618but these small molecules tend to suffer from fast clearance occasions and relatively small optical absorption cross-sections. Recently, Au-based nanostructures19,20and single-walled carbon nanotubes (SWCNTs)21have been exhibited as contrast brokers for PAT. Despite the impressive results, there is still ongoing debate around the toxicity of CNTs forin vivoapplications.2224In contrast, Au-based nanostructures have been approved by FDA as drug carriers or therapeutic agents for various phase-I clinical trials (e.g.,http://www.cytimmune.comandhttp://www.nanospectra.com). AuNCs symbolize an excellent candidate for serving as a photoacoustic (PA) contrast agent because of its strong and tunable light absorption in the NIR region, bio-inertness, easiness of bio-conjugation with a tumor-specific ligand, and the enhanced permeability and retention effect.2527 == RESULTS == The main objective of this study is to evaluate and quantitatively compare the PA contrast enhancement on B16 melanomas provided by both active and passive targeting AuNCs. In these cases, the surface of Azaperone the AuNCs was derivatized, respectively, with [Nle4, D-Phe7]–melanocyte-stimulating hormone ([Nle4, D-Phe7]–MSH-AuNCs) and poly(ethylene glycol) (PEG-AuNCs).25,28Figure 1ashows a schematic of the [Nle4, D-Phe7]–MSH- and PEG-AuNCs. The difference between these two AuNCs is Azaperone the presence of [Nle4, D-Phe7]–MSH at the distal end of PEG chain. [Nle4, D-Phe7]–MSH has been shown to strongly bind to -MSH receptors over-expressed on melanomas, and have higher resistance to enzymatic degradation and higher Azaperone specificity than the standard -MSH.29,30We focused on AuNCs with an outer edge length of 46 nm and a wall thickness of 7 nm (Fig. 1b). Upon surface modification, the optical resonance peak of the AuNCs was slightly red-shifted due to minor change in refractive index on the surface (Fig. 1c). The absorption cross-section of the AuNCs were measured as 1.71014m2and the ratio of absorption to extinction cross-sections was 0.7, confirming that this AuNCs absorb light very strongly (Supporting Information).26In a phantom, we could still detect PA signals from your AuNCs at a particle concentration of 4.5 pM and a signal-to-noise ratio (SNR) of 9 (Fig. 1dandSupporting Information). This detection limit.