Organic Alternatives to Quantum Dots for Intraoperative Near-Infrared Fluorescent Sentinel Lymph Node Mapping:

Intraoperative near-infrared (NIR) fluorescence imaging provides the surgeon with real-time image guidance during cancer and other surgeries. We have previously reported the use of NIR fluorescent quantum dots (QDs) for sentinel lymph node (SLN) mapping. However, because of concerns over potential toxicity, organic alternatives to QDs will be required for initial clinical studies. We describe a family of 800 nm organic heptamethine indocyanine-based contrast agents for SLN mapping spanning a spectrum from 775 Da small molecules to 7 MDa nanocolloids. We provide a detailed characterization of the optical and physical properties of these contrast agents and discuss the advantages and disadvantages of each. We present robust methods for the covalent conjugation, purification, and characterization of proteins with tetra-sulfonated heptamethine indocyanines, including mass spectroscopic site mapping of highly substituted molecules. One contrast agent, NIR fluorescent human serum albumin (HSA800), emerged as the molecule with the best overall performance with respect to entry to lymphatics, flow to the SLN, retention in the SLN, fluorescence yield and reproducibility. This preclinical study, performed on large animals approaching the size of humans, should serve as a foundation for future clinical studies. Mol Imaging (2005) 4, 172 – 181

Low-frequency wide-field fluorescence lifetime imaging using a high-power near-infrared light-emitting diode light source

Fluorescence lifetime imaging (FLi) could potentially improve exogenous near-infrared (NIR) fluorescence imaging, because it offers the capability of discriminating a signal of interest from background, provides real-time monitoring of a chemical environment, and permits the use of several different fluorescent dyes having the same emission wavelength. We present a high-power, LED-based, NIR light source for the clinical translation of wide-field (larger than 5 cm in diameter) FLi at frequencies up to 35 MHz. Lifetime imaging of indocyanine green (ICG), IRDye 800-CW, and 3,3′-diethylthiatricarbocyanine iodide (DTTCI) was performed over a large field of view (10 cm by 7.5 cm) using the LED light source. For comparison, a laser diode light source was employed as a gold standard. Experiments were performed both on the bench by diluting the fluorescent dyes in various chemical environments in Eppendorf tubes, and in vivo by injecting the fluorescent dyes mixed in Matrigel subcutaneously into CD-1 mice. Last, measured fluorescence lifetimes obtained using the LED and the laser diode sources were compared with those obtained using a state-of-the-art time-domain imaging system and with those previously described in the literature. On average, lifetime values obtained using the LED and the laser diode light sources were consistent, exhibiting a mean difference of 3% from the expected values and a coefficient of variation of 12%. Taken together, our study offers an alternative to laser diodes for clinical translation of FLi and explores the use of relatively low frequency modulation for in vivo imaging

Organic Alternatives to Quantum Dots for Intraoperative Near-Infrared Fluorescent Sentinel Lymph Node Mapping

Intraoperative near-infrared (NIR) fluorescence imaging provides the surgeon with real-time image guidance during cancer and other surgeries. We have previously reported the use of NIR fluorescent quantum dots (QDs) for sentinel lymph node (SLN) mapping. However, because of concerns over potential toxicity, organic alternatives to QDs will be required for initial clinical studies. We describe a family of 800 nm organic heptamethine indocyanine-based contrast agents for SLN mapping spanning a spectrum from 775 Da small molecules to 7 MDa nanocolloids. We provide a detailed characterization of the optical and physical properties of these contrast agents and discuss the advantages and disadvantages of each. We present robust methods for the covalent conjugation, purification, and characterization of proteins with tetra-sulfonated heptamethine indocyanines, including mass spectroscopic site mapping of highly substituted molecules. One contrast agent, NIR fluorescent human serum albumin (HSA800), emerged as the molecule with the best overall performance with respect to entry to lymphatics, flow to the SLN, retention in the SLN, fluorescence yield and reproducibility. This preclinical study, performed on large animals approaching the size of humans, should serve as a foundation for future clinical studies